Conjugated antisense compounds and their use

ABSTRACT

The present disclosure provides duplexes comprising a first oligomeric compound and a second oligomeric compound wherein the second oligomeric compound comprises a conjugate group. In certain embodiments, the duplex modulates the amount or activity of a target nucleic acid in extra hepatic tissues and/or extra hepatic cells. In certain embodiments, the duplex modulates the amount or activity of a target nucleic acid in hepatic tissues and/or hepatic cells.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCORE0137USC1SEQ_ST25.txt, created on Aug. 9, 2021, which is 8 Kb insize. The information in the electronic format of the sequence listingis incorporated herein by reference in its entirety.

FIELD

The present disclosure provides duplexes comprising a first oligomericcompound and a second oligomeric compound wherein the second oligomericcompound comprises a conjugate group. In certain embodiments, the duplexmodulates the amount or activity of a target nucleic acid in extrahepatic tissues and/or extra hepatic cells. In certain embodiments, theduplex modulates the amount or activity of a target nucleic acid inhepatic tissues and/or hepatic cells.

BACKGROUND

The principle behind antisense technology is that an antisense compoundhybridizes to a target nucleic acid and modulates the amount, activity,and/or function of the target nucleic acid. For example in certaininstances, antisense compounds result in altered transcription ortranslation of a target. Such modulation of expression can be achievedby, for example, target mRNA degradation or occupancy-based inhibition.An example of modulation of RNA target function by degradation is RNaseH-based degradation of the target RNA upon hybridization with a DNA-likeantisense compound. Another example of modulation of gene expression bytarget degradation is RNA interference (RNAi). RNAi refers toantisense-mediated gene silencing through a mechanism that utilizes theRNA-induced silencing complex (RISC). An additional example ofmodulation of RNA target function is by an occupancy-based mechanismsuch as is employed naturally by microRNA. MicroRNAs are smallnon-coding RNAs that regulate the expression of protein-coding RNAs. Thebinding of an antisense compound to a microRNA prevents that microRNAfrom binding to its messenger RNA targets, and thus interferes with thefunction of the microRNA. MicroRNA mimics can enhance native microRNAfunction. Certain antisense compounds alter splicing of pre-mRNA.Regardless of the specific mechanism, sequence-specificity makesantisense compounds attractive as tools for target validation and genefunctionalization, as well as therapeutics to selectively modulate theexpression of genes involved in the pathogenesis of diseases.

Antisense technology is an effective means for modulating the expressionof one or more specific gene products and can therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications. Chemically modified nucleosides may be incorporated intoantisense compounds to enhance one or more properties, such as nucleaseresistance, pharmacokinetics or affinity for a target nucleic acid. In1998, the antisense compound, Vitravene® (fomivirsen; developed by IsisPharmaceuticals Inc., Carlsbad, Calif.) was the first antisense drug toachieve marketing clearance from the U.S. Food and Drug Administration(FDA), and is currently a treatment of cytomegalovirus (CMV)-inducedretinitis in AIDS patients. For another example, an antisenseoligonucleotide targeting ApoB, KYNAMRO™, has been approved by the U.S.Food and Drug Administration (FDA) as an adjunct treatment tolipid-lowering medications and diet to reduce low densitylipoprotein-cholesterol (LDL-C), ApoB, total cholesterol (TC), andnon-high density lipoprotein-cholesterol (non HDL-C) in patients withhomozygous familial hypercholesterolemia (HoFH).

New chemical modifications have improved the potency and efficacy ofantisense compounds, uncovering the potential for oral delivery as wellas enhancing subcutaneous administration, decreasing potential for sideeffects, and leading to improvements in patient convenience. Chemicalmodifications increasing potency of antisense compounds allowadministration of lower doses, which reduces the potential for toxicity,as well as decreasing overall cost of therapy. Modifications increasingthe resistance to degradation result in slower clearance from the body,allowing for less frequent dosing. Different types of chemicalmodifications can be combined in one compound to further optimize thecompound's efficacy. Traditionally, antisense compounds, includingmodified oligonucleotides, have deomonstrated good functional uptakeinto liver tissue. However, there is still a need to facilitate uptakeand distribution of antisense compounds into other cell types.

SUMMARY OF THE INVENTION

After an oligomeric compound is administered to a subject, differentorgans, tissues, and cells receive different amounts of the oligomericcompound. The distribution of the oligomeric compound to differentorgans, tissues, and cells depends on many factors. For example, thedegree to which a given oligomeric compound binds to plasma proteins mayaffect the distribution of a given oligomeric compound to varioustissues. In certain embodiments, the degree to which a given oligomericcompound is recognized by certain cell-surface receptors may affect thedistribution of a given oligomeric compound to various tissues or cells.

Oligomeric compounds typically show good distribution to the liver afteradministration to a subject. However, in certain embodiments a needexists to deliver oligomeric compounds to other tissues within asubject. For example, a need exists to deliver oligomeric compounds toone or more extra-hepatic tissues such as adipose tissue or muscletissue. In certain embodiments, the present disclosure providesoligomeric compounds comprising a modified oligonucleotide and aconjugate group, wherein the conjugate group enhances delivery of themodified oligonucleotide to one or more extra-hepatic tissues.

In certain embodiments, the present disclosure provides a duplexcomprising a first oligomeric compound and a second oligomeric compound,wherein the second oligomeric compound comprises a modifiedoligonucleotide and a conjugate group. In certain embodiments, theduplex modulates the amount or activity of a target nucleic acidtranscript in an extra-hepatic cell to a greater extent than a duplexhaving a second oligomeric compound that does not comprise a conjugate.

The present disclosure provides the following non-limiting numberedembodiments:

-   Embodiment 1: A duplex comprising a first oligomeric compound and a    second oligomeric compound wherein    -   the first oligomeric compound comprises a first modified        oligonucleotide consisting of 10-30 linked nucleosides and has a        nucleobase sequence complementary to the nucleobase sequence of        the second oligomeric compound and to an extra-hepatic nucleic        acid target; and    -   the second oligomeric compound comprises a second modified        oligonucleotide consisting of 10-30 linked nucleosides and a        conjugate group;        -   wherein the conjugate group comprises a conjugate moiety and            a conjugate linker,        -   wherein the conjugate moiety is selected from among: a            lipid, vitamin, steroid, C₅-C₃₀ saturated alkyl group,            C₅-C₃₀ unsaturated alkyl group, fatty acid, and lipophilic            group;    -   and wherein the conjugate linker comprises at least one        cleavable moiety.-   Embodiment 2: A duplex comprising a first oligomeric compound and a    second oligomeric compound wherein    -   the first oligomeric compound comprises a first modified        oligonucleotide consisting of 10-30 linked nucleosides and has a        nucleobase sequence complementary to the nucleobase sequence of        the second oligomeric compound and to an extra-hepatic nucleic        acid target or a hepatic nucleic acid target; and    -   the second oligomeric compound comprises a second modified        oligonucleotide consisting of 10-30 linked nucleosides and a        conjugate group;        -   wherein the modified oligonucleotide has a sugar motif other            than:            -   1-4 2′-OMethyl-modified nucleosides at the 5′-end;            -   10-15 central nucleosides each comprising an unmodified                RNA sugar moiety; and            -   1-4 2′-OMethyl-modified nucleosides at the 3′-end;        -   wherein the conjugate group comprises a conjugate moiety and            a conjugate linker,        -   wherein the conjugate moiety is selected from among: a            lipid, vitamin, steroid, C₅-C₃₀ saturated alkyl group,            C₅-C₃₀ unsaturated alkyl group, fatty acid, and lipophilic            group;    -   and wherein the conjugate linker comprises at least one        cleavable moiety.-   Embodiment 3: A duplex comprising a first oligomeric compound and a    second oligomeric compound wherein    -   the first oligomeric compound comprises a first modified        oligonucleotide consisting of 10-30 linked nucleosides and has a        nucleobase sequence complementary to the nucleobase sequence of        the second oligomeric compound and to an extra-hepatic nucleic        acid target or a hepatic nucleic acid target; and    -   the second oligomeric compound comprises a second modified        oligonucleotide consisting of 10-30 linked nucleosides and a        conjugate group;        -   wherein the conjugate group comprises a conjugate moiety and            a conjugate linker,        -   wherein the conjugate moiety is selected from among: a            lipid, steroid, C₅-C₃₀ saturated alkyl group, C₅-C₃₀            unsaturated alkyl group, fatty acid, and a lipophilic group            other than a vitamin; and wherein the conjugate linker            comprises at least one cleavable moiety.-   Embodiment 4: The duplex of any of embodiments 1-3, wherein the    extra-hepatic nucleic acid target is not expressed in the liver at a    significant level.-   Embodiment 5: The duplex of any of embodiments 1-3, wherein the    extra-hepatic nucleic acid target is expressed in the liver at a    significant level.-   Embodiment 6: The duplex of any of embodiments 1-5, wherein the    extra-hepatic nucleic acid target is expressed in at least one    extra-hepatic cell type selected from among: white fat cells, brown    fat cells, adipocytes, macrophages, cancer cells, tumor cells,    smooth muscle cells, lymphocytes, heart muscle cells, and pulmonary    cells.-   Embodiment 7: The duplex of any of embodiments 1-6, wherein the    extra-hepatic nucleic acid target is expressed in at least two    extra-hepatic cell types.-   Embodiment 8: The duplex of any of embodiments 1-7, wherein the    extra-hepatic nucleic acid target is expressed in at least three    extra-hepatic cell types.-   Embodiment 9: The duplex of any of embodiments 1-8, wherein the    extra-hepatic nucleic acid target is expressed in at least four    extra-hepatic cell types.-   Embodiment 10: The duplex of any of embodiments 1-9, wherein the    extra-hepatic nucleic acid target is expressed in white fat cells.-   Embodiment 11: The duplex of any of embodiments 1-10, wherein the    extra-hepatic nucleic acid target is expressed in brown fat cells-   Embodiment 12: The duplex of any of embodiments 1-11, wherein the    extra-hepatic nucleic acid target is expressed in adipocytes.-   Embodiment 13: The duplex of any of embodiments 1-12, wherein the    extra-hepatic nucleic acid target is expressed in macrophages.-   Embodiment 14: The duplex of any of embodiments 1-13, wherein the    extra-hepatic nucleic acid target is expressed in cancer cells.-   Embodiment 15: The duplex of any of embodiments 1-14, wherein the    extra-hepatic nucleic acid target is expressed in tumor cells.-   Embodiment 16: The duplex of any of embodiments 1-15, wherein the    extra-hepatic nucleic acid target is expressed in smooth muscle    cells-   Embodiment 17: The duplex of any of embodiments 1-16, wherein the    extra-hepatic nucleic acid target is expressed in heart muscle    cells.-   Embodiment 18: The duplex of any of embodiments 1-17, wherein the    extra-hepatic nucleic acid target is expressed in lymphocytes.-   Embodiment 19: The duplex of any of embodiments 1-18, wherein the    extra-hepatic nucleic acid target is expressed in at least one    extra-hepatic tissue selected from among: skeletal muscle, cardiac    muscle, smooth muscle, adipose, white adipose, brown adipose,    spleen, bone, intestine, adrenal, testes, ovary, pancreas,    pituitary, prostate, skin, uterus, bladder, brain, glomerulus,    distal tubular epithelium, breast, lung, heart, kidney, ganglion,    frontal cortex, spinal cord, trigeminal ganglia, sciatic nerve,    dorsal root ganglion, epididymal fat, diaphragm, and colon.-   Embodiment 20: The duplex of any of embodiments 1-19, wherein the    extra-hepatic nucleic acid target is expressed in at least two    extra-hepatic tissues.-   Embodiment 21: The duplex of any of embodiments 1-20, wherein the    extra-hepatic nucleic acid target is expressed in at least three    extra-hepatic tissues.-   Embodiment 22: The duplex of any of embodiments 1-21, wherein the    extra-hepatic nucleic acid target is expressed in at least four    extra-hepatic tissues.-   Embodiment 23: The duplex of any of embodiments 1-22, wherein the    extra-hepatic nucleic acid target is expressed in skeletal muscle.-   Embodiment 24: The duplex of any of embodiments 1-23, wherein the    extra-hepatic nucleic acid target is expressed in cardiac muscle.-   Embodiment 25: The duplex of any of embodiments 1-24, wherein the    extra-hepatic nucleic acid target is expressed in smooth muscle.-   Embodiment 26: The duplex of any of embodiments 1-25, wherein the    extra-hepatic nucleic acid target is expressed in epididymal fat.-   Embodiment 27: The duplex of any of embodiments 1-26, wherein the    extra-hepatic nucleic acid target is expressed in white adipose    tissue.-   Embodiment 28: The duplex of any of embodiments 1-27, wherein the    extra-hepatic nucleic acid target is expressed in the spleen.-   Embodiment 29: The duplex of any of embodiments 1-28, wherein the    extra-hepatic nucleic acid target is expressed in bone.-   Embodiment 30: The duplex of any of embodiments 1-29, wherein the    extra-hepatic nucleic acid target is expressed in bone marrow.-   Embodiment 31: The duplex of any of embodiments 1-30, wherein the    extra-hepatic nucleic acid target is expressed in the intestine.-   Embodiment 32: The duplex of any of embodiments 1-31, wherein the    extra-hepatic nucleic acid target is expressed in adrenal tissue.-   Embodiment 33: The duplex of any of embodiments 1-32, wherein the    extra-hepatic nucleic acid target is expressed in the testes.-   Embodiment 34: The duplex of any of embodiments 1-33, wherein the    extra-hepatic nucleic acid target is expressed in the ovaries.-   Embodiment 35: The duplex of any of embodiments 1-34, wherein the    extra-hepatic nucleic acid target is expressed in the pancreas.-   Embodiment 36: The duplex of any of embodiments 1-35, wherein the    extra-hepatic nucleic acid target is expressed in the pituitary.-   Embodiment 37: The duplex of any of embodiments 1-36, wherein the    extra-hepatic nucleic acid target is expressed in the prostate.-   Embodiment 38: The duplex of any of embodiments 1-37, wherein the    extra-hepatic nucleic acid target is expressed in the skin.-   Embodiment 39: The duplex of any of embodiments 1-38, wherein the    extra-hepatic nucleic acid target is expressed in the uterus.-   Embodiment 40: The duplex of any of embodiments 1-39, wherein the    extra-hepatic nucleic acid target is expressed in the bladder.-   Embodiment 41: The duplex of any of embodiments 1-40, wherein the    extra-hepatic nucleic acid target is expressed in the brain.-   Embodiment 42: The duplex of any of embodiments 1-41, wherein the    extra-hepatic nucleic acid target is expressed in the glomerulus.-   Embodiment 43: The duplex of any of embodiments 1-42, wherein the    extra-hepatic nucleic acid target is expressed in the distal tubular    epithelium.-   Embodiment 44: The duplex of any of embodiments 1-43, wherein the    extra-hepatic nucleic acid target is expressed in the breast.-   Embodiment 45: The duplex of any of embodiments 1-44, wherein the    extra-hepatic nucleic acid target is expressed in the lung.-   Embodiment 46: The duplex of any of embodiments 1-45, wherein the    extra-hepatic nucleic acid target is expressed in the heart.-   Embodiment 47: The duplex of any of embodiments 1-46, wherein the    extra-hepatic nucleic acid target is expressed in the kidney.-   Embodiment 48: The duplex of any of embodiments 1-47, wherein the    extra-hepatic nucleic acid target is expressed in the colon.-   Embodiment 49: The duplex of any of embodiments 1-48, wherein the    extra-hepatic nucleic acid target is expressed in the ganglion.-   Embodiment 50: The duplex of any of embodiments 1-49, wherein the    extra-hepatic nucleic acid target is expressed in the frontal    cortex.-   Embodiment 51: The duplex of any of embodiments 1-50, wherein the    extra-hepatic nucleic acid target is expressed in the spinal cord.-   Embodiment 52: The duplex of any of embodiments 1-51, wherein the    extra-hepatic nucleic acid target is expressed in the trigeminal    ganglia.-   Embodiment 53: The duplex of any of embodiments 1-52, wherein the    extra-hepatic nucleic acid target is expressed in the sciatic nerve.-   Embodiment 54: The duplex of any of embodiments 1-53, wherein the    extra-hepatic nucleic acid target is expressed in the dorsal root    ganglion.-   Embodiment 55: The duplex of any of embodiments 1-54, wherein the    extra-hepatic nucleic acid target is an endogenous RNA transcript.-   Embodiment 56: The duplex of embodiment 55, wherein the RNA    transcript is a pre-mRNA.-   Embodiment 57: The duplex of embodiment 55, wherein the RNA    transcript is an mRNA.-   Embodiment 58: The duplex of embodiment 55, wherein the RNA    transcript is a toxic RNA.-   Embodiment 59: The duplex of embodiment 55, wherein the RNA    transcript is a non-coding RNA.-   Embodiment 60: The duplex of embodiment 55, wherein the RNA    transcript is a microRNA.-   Embodiment 61: The duplex of any of embodiments 1-54, wherein the    extra-hepatic nucleic acid target is viral nucleic acid.-   Embodiment 62: The duplex of any of embodiments 1-58, wherein the    extra-hepatic nucleic acid target is selected from among: ATGL,    CD40, TNF-α, CD36, DMPK, DNM2, DMD, DUX4, LMNA, ZFN9, SGLT2, and    GCCR.-   Embodiment 63: The duplex of any of embodiments 1-58, wherein the    extra-hepatic nucleic acid target is selected from among: Androgen    Receptor (AR), ANGPTL3, DGAT2, eIF4E, Factor XI, FGFR4, GCCR, GCGR,    GHR, PTP1B, SMRT, STAT3, Them1, TRPV4, FTO, MC4R, TMEM18, KCTD15,    GNPDA2, SH2B1, MTCH2, NEGR1, BDNF, ETV5, Leptin, leptin receptor,    FAIM2, KCNMA1, MAF, NRXN3, TFAP2B, MSRA, AGPAT2, BSCL2, AKT2, PPARγ,    LMNA, ZMPSTE24, DGAT1, TNFα, IL-6, Resistin, PAI-1, TBC1D1, METAP2,    VEGF, AIF-1, JNK1, CB1, RIP140, TIF2, ANGPT1, ANGPT2, EIF4EBP2,    CDK5, SLC13A5, Perilipin 1, Perilipin 2, Perilipin 3, Perilipin 4,    HGF, GDF3, TNKs, KATNA1, ChREBP, ATF4, BASP-1, NNMT.-   Embodiment 64: The duplex of any of embodiments 1-61, wherein the    extra-hepatic nucleic acid target is other than any of: Androgen    Receptor (AR), ANGPTL3, DGAT2, eIF4E, Factor XI, FGFR4, GCCR, GCGR,    GHR, PTP1B, SMRT, STAT3, Them1, TRPV4, FTO, MC4R, TMEM18, KCTD15,    GNPDA2, SH2B1, MTCH2, NEGR1, BDNF, ETV5, Leptin, leptin receptor,    FAIM2, KCNMA1, MAF, NRXN3, TFAP2B, MSRA, AGPAT2, BSCL2, AKT2, PPARγ,    LMNA, ZMPSTE24, DGAT1, TNFα, IL-6, Resistin, PAI-1, TBC1D1, METAP2,    VEGF, AIF-1, JNK1, CB1, RIP140, TIF2, ANGPT1, ANGPT2, EIF4EBP2,    CDK5, SLC13A5, Perilipin 1, Perilipin 2, Perilipin 3, Perilipin 4,    HGF, GDF3, TNKs, KATNA1, ChREBP, ATF4, BASP-1, NNMT.-   Embodiment 65: The duplex of any of embodiments 1-64, wherein the    first modified oligonucleotide has a nucleobase sequence that is at    least 80% complementary to the nucleobase sequence of the    extra-hepatic nucleic acid target, when measured across the entire    nucleobase sequence of the modified oligonucleotide.-   Embodiment 66: The duplex of embodiment 65, wherein the first    modified oligonucleotide has a nucleobase sequence that is at least    90% complementary to the nucleobase sequence of the extra-hepatic    nucleic acid target, when measured across the entire nucleobase    sequence of the modified oligonucleotide.-   Embodiment 67: The duplex of embodiment 65, wherein the first    modified oligonucleotide has a nucleobase sequence that is 100%    complementary to the nucleobase sequence of the extra-hepatic    nucleic acid target, when measured across the entire nucleobase    sequence of the modified oligonucleotide.-   Embodiment 68: The duplex of any of embodiments 1-55, wherein the    first modified oligonucleotide has at least 8 contiguous nucleobases    of any of the nucleobase sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6,    7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.-   Embodiment 69: The duplex of any of embodiments 1-55, wherein the    first modified oligonucleotide has at least 9 contiguous nucleobases    of any of the nucleobase sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6,    7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.-   Embodiment 70: The duplex of any of embodiments 1-55, wherein the    first modified oligonucleotide has at least 10 contiguous    nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1, 2,    3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,    22, or 23.-   Embodiment 71: The duplex of any of embodiments 1-55, wherein the    first modified oligonucleotide consists of the nucleobase sequence    of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,    15, 16, 17, 18, 19, 20, 21, 22, or 23.-   Embodiment 72: The duplex of any of embodiments 1-55, wherein the    first modified oligonucleotide has at least 12 contiguous    nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1, 2,    3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,    22, or 23.-   Embodiment 73: The duplex of any of embodiments 1-72, wherein the    first modified oligonucleotide does not have any    2′-deoxynucleosides.-   Embodiment 74: The duplex of any of embodiments 1-72, wherein the    first modified oligonucleotide comprises at least one modified    nucleoside.-   Embodiment 75: The duplex of embodiment 74, wherein the first    modified oligonucleotide comprises a least one modified nucleoside    comprising a modified sugar moiety.-   Embodiment 76: The duplex of embodiment 75, wherein the first    modified oligonucleotide comprises at least one modified nucleoside    comprising a bicyclic sugar moiety.-   Embodiment 77: The duplex of embodiment 76, wherein the first    modified oligonucleotide comprises at least one modified nucleoside    comprising a bicyclic sugar moiety having a 2′-4′ bridge, wherein    the 2′-4′ bridge is selected from —O—CH₂—; and —O—CH(CH₃)—.-   Embodiment 78: The duplex of any of embodiments 73-77, wherein the    first modified oligonucleotide comprises at least one modified    nucleoside comprising a modified non-bicyclic sugar moiety.-   Embodiment 79: The duplex of embodiment 78, wherein the first    modified oligonucleotide comprises at least one modified nucleoside    comprising a non-bicyclic sugar moiety comprising a 2′-MOE or    2′-O-Methyl modified sugar moiety.-   Embodiment 80: The duplex of any of embodiments 73-79, wherein the    first modified oligonucleotide comprises at least one modified    nucleoside comprising a sugar surrogate.-   Embodiment 81: The duplex of embodiment 80, wherein the first    modified oligonucleotide comprises at least one modified nucleoside    comprising a sugar surrogate selected from a morpholino, a PNA, a    F-HNA, a THP, or a modified THP.-   Embodiment 82: The duplex of any of embodiments 1-72 or 74-81,    wherein the first modified oligonucleotide comprises a sugar motif    having:    -   a 5′-region consisting of 1-5 linked 5′-nucleosides;    -   a central region consisting of 6-10 linked central region        nucleosides; and    -   a 3′-region consisting of 1-5 linked 3′-region nucleosides;        wherein    -   the nucleosides of the 5′-region, the 3′-region, and the central        region are contiguous, and the central region nucleosides each        comprise an unmodified DNA sugar moiety.-   Embodiment 83: The duplex of embodiment 82, wherein each of the    5′-region nucleosides and each of the 3′-region nucleosides comprise    a modified sugar moiety.-   Embodiment 84: The duplex of any of embodiments 1-83, wherein the    first modified oligonucleotide comprises at least one modified    internucleoside linkage.-   Embodiment 85: The duplex of embodiment 84, wherein each    internucleoside linkage of the first modified oligonucleotide is a    modified internucleoside linkage.-   Embodiment 86: The duplex of embodiment 84 or 85 wherein at least    one internucleoside linkage is a phosphorothioate internucleoside    linkage.-   Embodiment 87: The duplex of embodiment 84 or 86 wherein the first    modified oligonucleotide comprises at least one unmodified    phosphodiester internucleoside linkage.-   Embodiment 88: The duplex of embodiment 87, wherein each    internucleoside linkage is either an unmodified phosphodiester    internucleoside linkage or a phosphorothioate internucleoside    linkage.-   Embodiment 89: The duplex of embodiment 85, wherein each    internucleoside linkage is a phosphorothioate internucleoside    linkage.-   Embodiment 90: The duplex of any of embodiments 1-89, wherein the    first modified oligonucleotide comprises at least one modified    nucleobase.-   Embodiment 91: The duplex of embodiment 90, wherein the first    modified nucleobase is a 5-Methyl cytosine.-   Embodiment 92: The duplex of any of embodiments 1-91 wherein each    nucleobase of each nucleoside of the first modified oligonucleotide    is either an unmodified nucleobase or is 5-Methyl cytosine.-   Embodiment 93: The duplex of any of embodiments 1-92, wherein the    first modified oligonucleotide consists of 12-22 linked nucleosides.-   Embodiment 94: The duplex of any of embodiments 1-92, wherein the    first modified oligonucleotide consists of 12-20 linked nucleosides.-   Embodiment 95: The duplex of any of embodiments 1-92, wherein the    first modified oligonucleotide consists of 14-20 linked nucleosides.-   Embodiment 96: The duplex of any of embodiments 1-92, wherein the    first modified oligonucleotide consists of 16-20 linked nucleosides.-   Embodiment 97: The duplex of any of embodiments 1-92, wherein the    first modified oligonucleotide consists of 18-20 linked nucleosides.-   Embodiment 98: The duplex of any of embodiments 1-92, wherein the    first modified oligonucleotide consists of 20 linked nucleosides.-   Embodiment 99: The duplex of any of embodiments 1-92, wherein the    first modified oligonucleotide consists of 19 linked nucleosides.-   Embodiment 100: The duplex of any of embodiments 1-92, wherein the    first modified oligonucleotide consists of 18 linked nucleosides.-   Embodiment 101: The duplex of any of embodiments 1-92, wherein the    first modified oligonucleotide consists of 17 linked nucleosides.-   Embodiment 102: The duplex of any of embodiments 1-92, wherein the    first modified oligonucleotide consists of 16 linked nucleosides.-   Embodiment 103: The duplex of any of embodiments 1-102, wherein the    second modified oligonucleotide does not have any    2′deoxynucleosides.-   Embodiment 104: The duplex of any of embodiments 1-103, wherein the    second modified oligonucleotide comprises at least one modified    nucleoside.-   Embodiment 105: The duplex of embodiment 104, wherein the second    modified oligonucleotide comprises a least one modified nucleoside    comprising a modified sugar moiety.-   Embodiment 106: The duplex of embodiment 105, wherein the second    modified oligonucleotide comprises at least one modified nucleoside    comprising a bicyclic sugar moiety.-   Embodiment 107: The duplex of embodiment 106, wherein the second    modified oligonucleotide comprises at least one modified nucleoside    comprising a bicyclic sugar moiety having a 2′-4′ bridge, wherein    the 2′-4′ bridge is selected from —O—CH₂—; and —O—CH(CH₃)—.-   Embodiment 108: The duplex of any of embodiments 103-107, wherein    the second modified oligonucleotide comprises at least one modified    nucleoside comprising a modified non-bicyclic sugar moiety.-   Embodiment 109: The duplex of embodiment 108, wherein the first    modified oligonucleotide comprises at least one modified nucleoside    comprising a non-bicyclic sugar moiety comprising a 2′-MOE or    2′-O-Methyl modification.-   Embodiment 110: The duplex of any of embodiments 103-109 wherein the    second modified oligonucleotide does not comprise any nucleosides    comprising 2′-OMe.-   Embodiment 111: The duplex of any of embodiments 103-110, wherein    the second modified oligonucleotide comprises at least one modified    nucleoside comprising a sugar surrogate.-   Embodiment 112: The duplex of embodiment 111, wherein the second    modified oligonucleotide comprises at least one modified nucleoside    comprising a sugar surrogate selected from a morpholino, a PNA, a    F-HNA, a THP, or a modified THP.-   Embodiment 113: The duplex of any of embodiments 104-112, wherein    the second modified oligonucleotide has a sugar motif comprising:    -   a 5′-region consisting of 1-5 linked 5′-nucleosides;    -   a central region consisting of 6-10 linked central region        nucleosides; and    -   a 3′-region consisting of 1-5 linked 3′-region nucleosides;        wherein    -   the nucleosides of the 5′-region, the 3′-region, and the central        region are contiguous; and the central region nucleosides each        comprise an unmodified 2′-deoxy sugar moiety.-   Embodiment 114: The duplex of embodiment 113 wherein each of the    5′-region nucleosides and each of the 3′-region nucleosides comprise    a modified sugar moiety.-   Embodiment 115: The duplex of embodiment 114, wherein the 5′-region    nucleosides and the 3′-region nucleosides of the second modified    oligonucleotide are selected from: F-RNA modified nucleosides,    2′-MOE modified nucleosides, LNA nucleosides, and cEt nucleosides.-   Embodiment 116: The duplex of any of embodiments 104-112 wherein the    second modified oligonucleotide has sugar motif comprising    alternating 2′-deoxynucleosides and 2′-MOE modified nucleosides.-   Embodiment 117: The duplex of any of embodiments 103-116, wherein    the second modified oligonucleotide comprises at least one modified    internucleoside linkage.-   Embodiment 118: The duplex of embodiment 117, wherein each    internucleoside linkage of the second modified oligonucleotide is a    modified internucleoside linkage.-   Embodiment 119: The duplex of embodiment 117 or 118 wherein at least    one internucleoside linkage of the second oligonucleotide is a    phosphorothioate internucleoside linkage.-   Embodiment 120: The duplex of embodiment 117 or 119 wherein the    second modified oligonucleotide comprises at least one unmodified    phosphodiester internucleoside linkage.-   Embodiment 121: The duplex of embodiment 120, wherein each    internucleoside linkage of the second oligonucleotide is either an    unmodified phosphodiester internucleoside linkage or a    phosphorothioate internucleoside linkage.-   Embodiment 122: The duplex of embodiment 118, wherein each    internucleoside linkage of the second oligonucleotide is a    phosphorothioate internucleoside linkage.-   Embodiment 123: The duplex of any of embodiments 117 or 119-121    wherein the second oligonucleotide has a center region comprising    unmodified phosphodiester internucleoside linkages.-   Embodiment 124: The duplex of embodiment 123, wherein the second    oligonucleotide has 1 or 2 terminal phosphorothioate linkages on one    end or on both ends.-   Embodiment 125: The duplex of any of embodiments 103-124, wherein    the second modified oligonucleotide comprises at least one modified    nucleobase.-   Embodiment 126: The duplex of embodiment 125, wherein the second    modified nucleobase is a 5-Methyl cytosine.-   Embodiment 127: The duplex of any of embodiments 103-126 wherein    each nucleobase of each nucleoside of the second modified    oligonucleotide is either an unmodified nucleobase or is 5-Methyl    cytosine.-   Embodiment 128: The duplex of any of embodiments 103-127, wherein    the second modified oligonucleotide consists of 12-22 linked    nucleosides.-   Embodiment 129: The duplex of any of embodiments 103-127, wherein    the second modified oligonucleotide consists of 12-20 linked    nucleosides.-   Embodiment 130: The duplex of any of embodiments 103-127, wherein    the second modified oligonucleotide consists of 14-20 linked    nucleosides.-   Embodiment 131: The duplex of any of embodiments 103-127, wherein    the second modified oligonucleotide consists of 16-20 linked    nucleosides.-   Embodiment 132: The duplex of any of embodiments 103-127, wherein    the second modified oligonucleotide consists of 18-20 linked    nucleosides.-   Embodiment 133: The duplex of any of embodiments 103-127, wherein    the second modified oligonucleotide consists of 20 linked    nucleosides.-   Embodiment 134: The duplex of any of embodiments 103-127, wherein    the second modified oligonucleotide consists of 19 linked    nucleosides.-   Embodiment 135: The duplex of any of embodiments 103-127, wherein    the second modified oligonucleotide consists of 18 linked    nucleosides.-   Embodiment 136: The duplex of any of embodiments 103-127, wherein    the second modified oligonucleotide consists of 17 linked    nucleosides.-   Embodiment 137: The duplex of any of embodiments 103-127, wherein    the second modified oligonucleotide consists of 16 linked    nucleosides.-   Embodiment 138: The duplex of any of embodiments 1-137, wherein the    conjugate linker comprises 1-5 linker-nucleosides.-   Embodiment 139: The duplex of embodiment 138, wherein the conjugate    linker comprises 3 linker-nucleosides.-   Embodiment 140: The duplex of embodiment 139, wherein the 3    linker-nucleosides have a TCA motif.-   Embodiment 141: The duplex of embodiment 138, wherein 1-5    linker-nucleosides do not comprise a TCA motif.-   Embodiment 142: The duplex of any of embodiments 1-137, wherein the    conjugate group does not comprise linker-nucleosides.-   Embodiment 143: The duplex of any of embodiments 1-142, wherein the    conjugate linker comprises a hexylamino group.-   Embodiment 144: The duplex of any of embodiments 1-143, wherein the    conjugate linker comprises a polyethylene glycol group.-   Embodiment 145: The duplex of any of embodiments 1-144, wherein the    conjugate linker comprises a triethylene glycol group.-   Embodiment 146: The duplex of any of embodiments 1-145, wherein the    conjugate linker comprises a phosphate group.-   Embodiment 147: The duplex of any of embodiments 1-146, wherein the    conjugate linker comprises:

-   -   X directly or indirectly attaches to the conjugate moiety; and    -   Y directly or indirectly attaches to the second modified        oligonucleotide.

-   Embodiment 148: The duplex of embodiment 147, wherein X comprises 0.

-   Embodiment 149: The duplex of embodiment 147 or 148, wherein Y    comprises a phosphate group.

-   Embodiment 150: The duplex of any of embodiments 1-146, wherein the    conjugate linker comprises:

-   -   X directly or indirectly attaches to the conjugate moiety; and    -   T₁ comprises a linking group, nucleoside, or a modified        oligonucleotide.

-   Embodiment 151: The duplex of any of embodiments 1-146, wherein the    conjugate linker comprises:

-   -   X directly or indirectly attaches to the conjugate moiety; and    -   wherein T₁ comprises a nucleotide or a modified oligonucleotide;        and B_(x) is a modified or unmodified nucleobase.

-   Embodiment 152: The duplex of any of embodiments 1-151, wherein the    conjugate moiety comprises lipophilic group.

-   Embodiment 153: The duplex of embodiment 152, wherein the lipophilic    group is selected from among: cholesterol, C₁₀-C₂₆ saturated fatty    acid, C₁₀-C₂₆ unsaturated fatty acid, C₁₀-C₂₆ alkyl, triglyceride,    tocopherol, or cholic acid.

-   Embodiment 154: The duplex of embodiment 153, wherein the conjugate    moiety is a saturated fatty acid or an unsaturated fatty acid.

-   Embodiment 155: The duplex of embodiment 153, wherein the conjugate    moiety is C₁₆ lipid.

-   Embodiment 156: The duplex of embodiment 153, wherein the conjugate    moiety is C₁₈ lipid.

-   Embodiment 157: The duplex of embodiment 153, wherein the conjugate    moiety is C₁₆ alkyl.

-   Embodiment 158: The duplex of embodiment 153, wherein the conjugate    moiety is C₁₈ alkyl.

-   Embodiment 159: The duplex of embodiment 153, wherein the conjugate    moiety is cholesterol.

-   Embodiment 160: The duplex of embodiment 153, wherein the conjugate    moiety is tocopherol.

-   Embodiment 161: The duplex of any of embodiments 1-160, wherein the    conjugate group is attached to the second modified oligonucleotide    at the 5′-end of the second modified oligonucleotide.

-   Embodiment 162: The duplex of any of embodiments 1-160, wherein the    conjugate group is attached to the second modified oligonucleotide    at the 3′-end of the second modified oligonucleotide.

-   Embodiment 163: The duplex of any of embodiments 1-162 comprising a    terminal group.

-   Embodiment 164: An antisense compound consisting of the duplex of    any of embodiments 1-163.

-   Embodiment 165: An antisense compound comprising the duplex of any    of embodiments 1-163.

-   Embodiment 166: The antisense compound of embodiment 164 or 165 that    is an RNase H antisense compound.

-   Embodiment 167: The antisense compound of embodiment 164 or 165 that    is an RNAi antisense compound.

-   Embodiment 168: The antisense compound of any of embodiments 164-167    that is capable of reducing the amount or activity of the    extra-hepatic nucleic acid target by at least 20% when tested at a    concentration of 1.0 nM in a standard cell assay.

-   Embodiment 169: The antisense compound of embodiment 168 that is    capable of reducing the amount or activity of the extra-hepatic    nucleic acid target by at least 40% in the standard cell assay.

-   Embodiment 170: The antisense compound of embodiment 168 that is    capable of reducing the amount or activity of the extra-hepatic    nucleic acid target by at least 80% in the standard cell assay.

-   Embodiment 171: The antisense compound of any of embodiments 164-170    that is capable of reducing the amount or activity of the    extra-hepatic nucleic acid target in an extra-hepatic tissue by at    least 20% when provided at a dose of 100 mg/kg in a standard animal    experiment.

-   Embodiment 172: The antisense compound of embodiment 171 that is    capable of reducing the amount or activity of the extra-hepatic    nucleic acid target in the extra-hepatic tissue by at least 40%.

-   Embodiment 173: The antisense compound of embodiment 171 that is    capable of reducing the amount or activity of the extra-hepatic    nucleic acid target in the extra-hepatic tissue by at least 80%.

-   Embodiment 174: The antisense compound of embodiment 164 or 165 that    alters the RNA processing of the extra-hepatic nucleic acid target.

-   Embodiment 175: A method comprising contacting a cell with the    duplex of any of embodiments 1-163.

-   Embodiment 176: A method comprising contacting a cell with the    antisense compound of any of embodiments 164-174.

-   Embodiment 177: A method of modulating the amount or activity of an    extra-hepatic nucleic acid target in a cell comprising contacting    the cell with the duplex or antisense compound of any of embodiments    1-174 and thereby modulating the amount or activity of the    extra-hepatic nucleic acid target in the cell.

-   Embodiment 178: The method of embodiment 177, wherein the amount or    activity of the extra-hepatic nucleic acid target is reduced.

-   Embodiment 179: The method of any of embodiments 175-178, wherein    the cell is in vitro.

-   Embodiment 180: The method of any of embodiments 175-178, wherein    the cell is in an animal.

-   Embodiment 181: The method of embodiment 180, wherein the animal is    a human.

-   Embodiment 182: A method of modulating the amount or activity of a    hepatic nucleic acid target in a cell comprising contacting the cell    with the duplex or antisense compound of any of embodiments 1-174    and thereby modulating the amount or activity of the hepatic nucleic    acid target in the cell.

-   Embodiment 183: The method of embodiment 182, wherein the amount or    activity of the extra-hepatic nucleic acid target is reduced.

-   Embodiment 184: The method of embodiment 182 or 183, wherein the    cell is in vitro.

-   Embodiment 185: The method of embodiment 182 or 183, wherein the    cell is in an animal.

-   Embodiment 186: The method of embodiment 185, wherein the animal is    a human.

-   Embodiment 187: A pharmaceutical composition comprising a duplex of    any embodiments 1-163 and a pharmaceutically acceptable carrier or    diluent.

-   Embodiment 188: A pharmaceutical composition comprising an antisense    compound of any of embodiments 164-174 and a pharmaceutically    acceptable carrier or diluent.

-   Embodiment 189: A method comprising administering to an animal a    pharmaceutical composition of embodiment 187 or 188.

-   Embodiment 190: A method of treating a disease associated with an    extra-hepatic nucleic acid target comprising administering to an    individual having or at risk for developing a disease associated    with the extra-hepatic nucleic acid target a therapeutically    effective amount of a pharmaceutical composition according to    embodiment 187 or 188; and thereby treating the disease associated    with the extra-hepatic nucleic acid target.

-   Embodiment 191: The method of embodiment 190, wherein the    extra-hepatic nucleic acid target is selected from among: ATGL,    CD40, CD36, DMPK, DNM2, DMD, DUX4, LMNA, ZFN9, SGLT2, or GCCR.

-   Embodiment 192: The method of embodiment 190, wherein the    extra-hepatic nucleic acid target transcript is selected from among:    Androgen Receptor (AR), ANGPTL3, DGAT2, eIF4E, Factor XI, FGFR4,    GCCR, GCGR, GHR, PTP1B, SMRT, STAT3, Them1, TRPV4, FTO, MC4R,    TMEM18, KCTD15, GNPDA2, SH2B1, MTCH2, NEGR1, BDNF, ETV5, Leptin,    leptin receptor, FAIM2, KCNMA1, MAF, NRXN3, TFAP2B, MSRA, AGPAT2,    BSCL2, AKT2, PPARγ, LMNA, ZMPSTE24, DGAT1, TNFα, IL-6, Resistin,    PAI-1, TBC1D1, METAP2, VEGF, AIF-1, JNK1, CB1, RIP140, TIF2, ANGPT1,    ANGPT2, EIF4EBP2, CDK5, SLC13A5, Perilipin 1, Perilipin 2, Perilipin    3, Perilipin 4, HGF, GDF3, TNKs, KATNA1, ChREBP, ATF4, BASP-1, NNMT.

-   Embodiment 193: The method of embodiment 190, wherein the    extra-hepatic nucleic acid target transcript is not selected from    among: Androgen Receptor (AR), ANGPTL3, DGAT2, eIF4E, Factor XI,    FGFR4, GCCR, GCGR, GHR, PTP1B, SMRT, STAT3, Them1, TRPV4, FTO, MC4R,    TMEM18, KCTD15, GNPDA2, SH2B1, MTCH2, NEGR1, BDNF, ETV5, Leptin,    leptin receptor, FAIM2, KCNMA1, MAF, NRXN3, TFAP2B, MSRA, AGPAT2,    BSCL2, AKT2, PPARγ, LMNA, ZMPSTE24, DGAT1, TNFα, IL-6, Resistin,    PAI-1, TBC1D1, METAP2, VEGF, AIF-1, JNK1, CB1, RIP140, TIF2, ANGPT1,    ANGPT2, EIF4EBP2, CDK5, SLC13A5, Perilipin 1, Perilipin 2, Perilipin    3, Perilipin 4, HGF, GDF3, TNKs, KATNA1, ChREBP, ATF4, BASP-1, NNMT.

-   Embodiment 194: The method of any of embodiments 190-193, wherein at    least one symptom of a disease associated with an extra-hepatic    nucleic acid target is ameliorated.

-   Embodiment 195: The method of any of embodiments 190-194, wherein    the disease is selected from among: diabetes, metabolic syndrome,    cardiac disease, muscular dystrophy, myotonic dystrophy, Becker    muscular dystrophy, congenital muscular dystrophy, Duchenne muscular    dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular    dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle    muscular dystrophy, or oculopharyngeal muscular dystrophy.

-   Embodiment 196: The method of any of embodiments 189-195 wherein the    amount or activity of the extra-hepatic nucleic acid target is    modulated in at least one tissue type other than liver.

-   Embodiment 197: The method of embodiment 196, wherein the amount of    activity of the extra-hepatic nucleic acid target is modulated in at    least two tissue types.

-   Embodiment 198: The method of embodiment 197, wherein at least one    of the at least two tissue types is selected from among: liver,    skeletal muscle, cardiac muscle, smooth muscle, adipose, white    adipose, spleen, bone, intestine, adrenal, testes, ovary, pancreas,    pituitary, prostate, skin, uterus, bladder, brain, glomerulus,    distal tubular epithelium, breast, lung, heart, kidney, ganglion,    frontal cortex, spinal cord, trigeminal ganglia, sciatic nerve,    dorsal root ganglion, epididymal fat, diaphragm, and colon.

-   Embodiment 199: The method of embodiment 198, wherein at least two    tissue types are selected from among: liver, skeletal muscle,    cardiac muscle, smooth muscle, adipose, white adipose, spleen, bone,    intestine, adrenal, testes, ovary, pancreas, pituitary, prostate,    skin, uterus, bladder, brain, glomerulus, distal tubular epithelium,    breast, lung, heart, kidney, ganglion, frontal cortex, spinal cord,    trigeminal ganglia, sciatic nerve, dorsal root ganglion, epididymal    fat, diaphragm, and colon.

-   Embodiment 200: A method of treating a multi-tissue disease or    condition, comprising administering a therapeutically effective    amount of the pharmaceutical composition of embodiment 187 or 188 to    a subject, and thereby modulating the amount or activity of a target    nucleic acid in two or more tissues.

-   Embodiment 201: A method of treating a disease or condition,    comprising administering a therapeutically effective amount of the    pharmaceutical composition of embodiment 187 or 188 to a subject,    and thereby modulating the amount or activity of a target nucleic    acid in two or more cell types.

-   Embodiment 202: A method of treating a multi-tissue disease or    condition, comprising administering a therapeutically effective    amount of the pharmaceutical composition of embodiment 187 or 188 to    a subject, and thereby modulating the amount or activity of a target    nucleic acid in two or more cell types.

-   Embodiment 203: The method of embodiment 201 or 202, wherein the two    or more cell types are selected from among: hepatocytes, white fat    cells, brown fat cells, adipocytes, macrophages, cancer cells, tumor    cells, smooth muscle cells, lymphocytes, and heart muscle cells.

-   Embodiment 204: The method of any of embodiments 189-203, wherein    the pharmaceutical composition is administered subcutaneously.

-   Embodiment 205: The method of any of embodiments 189-203, wherein    the pharmaceutical composition is administered intravenously.

-   Embodiment 206: The method of any of embodiments 189-203, wherein    the pharmaceutical composition is administered by parenteral    administration.

-   Embodiment 207: The method of any of embodiments 189-203, wherein    the pharmaceutical composition is administered by intraperitoneal    administration.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive. Herein, the use of the singular includes theplural unless specifically stated otherwise. As used herein, the use of“or” means “and/or” unless stated otherwise. Furthermore, the use of theterm “including” as well as other forms, such as “includes” and“included”, is not limiting. Also, terms such as “element” or“component” encompass both elements and components comprising one unitand elements and components that comprise more than one subunit, unlessspecifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated-by-reference forthe portions of the document discussed herein, as well as in theirentirety.

Definitions

Unless specific definitions are provided, the nomenclature used inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Where permitted, all patents, applications, published applicationsand other publications and other data referred to throughout in thedisclosure are incorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

“2′-deoxynucleoside” means a nucleoside comprising 2′-H(H) furanosylsugar moiety, as found in naturally occurring deoxyribonucleic acids(DNA). In certain embodiments, a 2′-deoxynucleoside may comprise amodified nucleobase or may comprise an RNA nucleobase (uracil).

“2′-substituted nucleoside” or “2-modified nucleoside” means anucleoside comprising a 2′-substituted or 2′-modified sugar moiety. Asused herein, “2′-substituted” or “2-modified” in reference to a sugarmoiety means a sugar moiety comprising at least one 2′-substituent groupother than H or OH.

“Antisense activity” means any detectable and/or measurable changeattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein encodedby such target nucleic acid compared to target nucleic acid levels ortarget protein levels in the absence of the antisense compound. Incertain embodiments, antisense activity is a change in splicing of apre-mRNA nucleic acid target. In certain embodiments, antisense activityis an increase in the amount or expression of a target nucleic acid orprotein encoded by such target nucleic acid compared to target nucleicacid levels or target protein levels in the absence of the antisensecompound.

“Antisense compound” means a compound comprising an antisenseoligonucleotide and optionally one or more additional features, such asa conjugate group or terminal group.

“Antisense oligonucleotide” means an oligonucleotide that (1) has anucleobase sequence that is at least partially complementary to a targetnucleic acid and that (2) is capable of producing an antisense activityin a cell or animal.

“Ameliorate” in reference to a treatment means improvement in at leastone symptom relative to the same symptom in the absence of thetreatment. In certain embodiments, amelioration is the reduction in theseverity or frequency of a symptom or the delayed onset or slowing ofprogression in the severity or frequency of a symptom.

“Bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclicsugar moiety. As used herein, “bicyclic sugar” or “bicyclic sugarmoiety” means a modified sugar moiety comprising two rings, wherein thesecond ring is formed via a bridge connecting two of the atoms in thefirst ring thereby forming a bicyclic structure. In certain embodiments,the first ring of the bicyclic sugar moiety is a furanosyl moiety. Incertain embodiments, the bicyclic sugar moiety does not comprise afuranosyl moiety.

“Branching group” means a group of atoms having at least 3 positionsthat are capable of forming covalent linkages to at least 3 groups. Incertain embodiments, a branching group provides a plurality of reactivesites for connecting tethered ligands to an oligonucleotide via aconjugate linker and/or a cleavable moiety.

“Cell-targeting moiety” means a conjugate group or portion of aconjugate group that is capable of binding to a particular cell type orparticular cell types.

“Cleavable moiety” means a bond or group of atoms that is cleaved underphysiological conditions, for example, inside a cell, an animal, or ahuman.

“Complementary” in reference to an oligonucleotide means that at least70% of the nucleobases of such oligonucleotide or one or more regionsthereof and the nucleobases of another nucleic acid or one or moreregions thereof are capable of hydrogen bonding with one another whenthe nucleobase sequence of the oligonucleotide and the other nucleicacid are aligned in opposing directions. Complementary nucleobases meansnucleobases that are capable of forming hydrogen bonds with one another.Complementary nucleobase pairs include, but unless otherwise specificare not limited to, adenine (A) and thymine (T), adenine (A) and uracil(U), cytosine (C) and guanine (G), 5-methyl cytosine (mC) and guanine(G). Complementary oligonucleotides and/or nucleic acids need not havenucleobase complementarity at each nucleoside. Rather, some mismatchesare tolerated. As used herein, “fully complementary” or “100%complementary” in reference to oligonucleotides means that sucholigonucleotides are complementary to another oligonucleotide or nucleicacid at each nucleoside of the oligonucleotide.

“Conjugate group” means a group of atoms that is directly or indirectlyattached to an oligonucleotide. Conjugate groups include a conjugatemoiety and a conjugate linker that attaches the conjugate moiety to theoligonucleotide.

“Conjugate linker” means a group of atoms comprising at least one bondthat connects a conjugate moiety to an oligonucleotide.

“Conjugate moiety” means a group of atoms that is attached to anoligonucleotide via a conjugate linker.

“Contiguous” in the context of an oligonucleotide refers to nucleosides,nucleobases, sugar moieties, or internucleoside linkages that areimmediately adjacent to each other. For example, “contiguousnucleobases” means nucleobases that are immediately adjacent to eachother in a sequence.

“Duplex” means two oligomeric compounds that are paired. In certainembodiments, the two oligomeric compounds are paired via hybridizationof complementary nucleobases.

“Extra-hepatic cell type” means a cell type that is not a hepatocyte.

“Extra-hepatic nucleic acid target” means a target nucleic acid that isexpressed in tissues other than liver. In certain embodiments,extra-hepatic nucleic acid targets are not expressed in the liver or notexpressed in the liver at a significant level. In certain embodiments,extra-hepatic nucleic acid targets are expressed outside the liver andalso in the liver.

“Extra-hepatic tissue” means a tissue other than liver.

“Fully modified” in reference to a modified oligonucleotide means amodified oligonucleotide in which each sugar moiety is modified.“Uniformly modified” in reference to a modified oligonucleotide means afully modified oligonucleotide in which each sugar moiety is the same.For example, the nucleosides of a uniformly modified oligonucleotide caneach have a 2′-MOE modification but different nucleobase modifications,and the internucleoside linkages may be different.

“Gapmer” means an antisense oligonucleotide comprising an internalregion having a plurality of nucleosides that support RNase H cleavagepositioned between external regions having one or more nucleosides,wherein the nucleosides comprising the internal region are chemicallydistinct from the nucleoside or nucleosides comprising the externalregions. The internal region may be referred to as the “gap” and theexternal regions may be referred to as the “wings.”

“Hybridization” means the pairing or annealing of complementaryoligonucleotides and/or nucleic acids. While not limited to a particularmechanism, the most common mechanism of hybridization involves hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleobases.

“Inhibiting the expression or activity” refers to a reduction orblockade of the expression or activity relative to the expression ofactivity in an untreated or control sample and does not necessarilyindicate a total elimination of expression or activity.

“Internucleoside linkage” means a group or bond that forms a covalentlinkage between adjacent nucleosides in an oligonucleotide. As usedherein “modified internucleoside linkage” means any internucleosidelinkage other than a naturally occurring, phosphate internucleosidelinkage. Non-phosphate linkages are referred to herein as modifiedinternucleoside linkages. “Phosphorothioate linkage” means a modifiedphosphate linkage in which one of the non-bridging oxygen atoms isreplaced with a sulfur atom. A phosphorothioate internucleoside linkageis a modified internucleoside linkage.

“Linker-nucleoside” means a nucleoside that links, either directly orindirectly, an oligonucleotide to a conjugate moiety. Linker-nucleosidesare located within the conjugate linker of an oligomeric compound.Linker-nucleosides are not considered part of the oligonucleotideportion of an oligomeric compound even if they are contiguous with theoligonucleotide.

“Lipophilic group” or “lipophilic” in reference to a chemical groupmeans a group of atoms that is more soluble in lipids or organicsolvents than in water and/or has a higher affinity for lipids than forwater. In certain embodiments, lipophilic groups comprise a lipid. Asused herein “lipid” means a molecule that is not soluble in water or isless soluble in water than in organic solvents. In certain embodiments,compounds of the present invention comprise lipids selected fromsaturated or unsaturated fatty acids, steroids, fat soluble vitamins,phospholipids, sphingolipids, hydrocarbons, mono-, di-, andtri-glycerides, and synthetic derivatives thereof.

“Non-bicyclic modified sugar” or “non-bicyclic modified sugar moiety”means a modified sugar moiety that comprises a modification, such as asubstitutent, that does not form a bridge between two atoms of the sugarto form a second ring.

“Linked nucleosides” are nucleosides that are connected in a continuoussequence (i.e. no additional nucleosides are present between those thatare linked).

“Mismatch” or “non-complementary” means a nucleobase of a firstoligonucleotide that is not complementary with the correspondingnucleobase of a second oligonucleotide or target nucleic acid when thefirst and second oligomeric compound are aligned.

“MOE” means methoxyethyl. “2′-MOE” means a —OCH₂CH₂OCH₃ group at the 2′position of a furanosyl ring.

“Motif” means the pattern of unmodified and/or modified sugar moieties,nucleobases, and/or internucleoside linkages, in an oligonucleotide.

“Multi-tissue disease or condition” means a disease or condition affectsor is effected by more than one tissue. In treating a multi-tissuedisease or condition, it is desirable to affect more than one tissuetype. In certain embodiments, treatment of disease or condition may beenhanced by treating the disease or condition in multiple tissues. Forexample, in certain embodiments, a disease or condition may manifestitself in the liver tissue and the muscle tissue. In certainembodiments, treating the disease or condition in the liver tissue andthe muscle tissue will be more effective than treating the disease ineither the liver tissue or the muscle tissue.

“Naturally occurring” means found in nature.

“Nucleobase” means an unmodifiednucleobase or a modified nucleobase. Asused herein a “an “unmodified nucleobase” is adenine (A), thymine (T),cytosine (C), uracil (U), and guanine (G). As used herein, a “modifiednucleobase” is a group of atoms other than unmodified A, T, C, U, or Gcapable of pairing with at least one unmodified nucleobase. A universalbase is a modified nucleobase that can pair with any one of the fiveunmodified nucleobases. As used herein, “nucleobase sequence” means theorder of contiguous nucleobases in a nucleic acid or oligonucleotideindependent of any sugar or internucleoside linkage modification.

“Nucleoside” means a compound comprising a nucleobase and a sugarmoiety. The nucleobase and sugar moiety are each, independently,unmodified or modified. As used herein, “modified nucleoside” means anucleoside comprising a modified nucleobase and/or a modified sugarmoiety. Modified nucleosides include abasic nucleosides, which lack anucleobase.

“Oligomeric compound” means a compound consisting of an oligonucleotideand optionally one or more additional features, such as a conjugategroup or terminal group.

“Oligonucleotide” means a strand of linked nucleosides connected viainternucleoside linkages, wherein each nucleoside and internucleosidelinkage may be modified or unmodified. Unless otherwise indicated,oligonucleotides consist of 8-50 linked nucleosides. As used herein,“modified oligonucleotide” means an oligonucleotide, wherein at leastone nucleoside or internucleoside linkage is modified. As used herein,“unmodified oligonucleotide” means an oligonucleotide that does notcomprise any nucleoside modifications or internucleoside modifications.

“Pharmaceutically acceptable carrier or diluent” means any substancesuitable for use in administering to an animal. Certain such carriersenable pharmaceutical compositions to be formulated as, for example,tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspension and lozenges for the oral ingestion by a subject. In certainembodiments, a pharmaceutically acceptable carrier or diluent is sterilewater; sterile saline; or sterile buffer solution.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of compounds, such as oligomericcompounds, i.e., salts that retain the desired biological activity ofthe parent compound and do not impart undesired toxicological effectsthereto.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to a subject. For example, a pharmaceutical compositionmay comprise an antisense compound and a sterile aqueous solution. Incertain embodiments, a pharmaceutical composition shows activity in freeuptake assay in certain cell lines.

“Phosphorus moiety” means a group of atoms comprising a phosphorus atom.In certain embodiments, a phosphorus moiety comprises a mono-, di-, ortri-phosphate, or phosphorothioate.

“Prodrug” means a therapeutic agent in a form outside the body that isconverted to a different form within the body or cells thereof.Typically conversion of a prodrug within the body is facilitated by theaction of an enzymes (e.g., endogenous or viral enzyme) or chemicalspresent in cells or tissues and/or by physiologic conditions.

“RNAi compound” means an antisense compound that acts, at least in part,through RISC or Ago2 to modulate a target nucleic acid and/or proteinencoded by a target nucleic acid. RNAi compounds include, but are notlimited to double-stranded siRNA, single-stranded RNA (ssRNA), andmicroRNA, including microRNA mimics. In certain embodiments, an RNAicompound modulates the amount, activity, and/or splicing of a targetnucleic acid. The term RNAi compound excludes antisense oligonucleotidesthat act through RNase H.

“Single-stranded” in reference to an oligomeric compound means such acompound that is not paired with a second oligomeric compound to form aduplex. “Self-complementary” in reference to an oligonucleotide means anoligonucleotide that at least partially hybridizes to itself. A compoundconsisting of one oligomeric compound, wherein the oligonucleotide ofthe oligomeric compound is self-complementary, is a single-strandedcompound. A single-stranded antisense or oligomeric compound may becapable of binding to a complementary oligomeric compound to form aduplex, in which case it would no longer be single-stranded.

“Standard cell assay” means the assay described in Example 1 andreasonable variations thereof

“Standard in vivo experiment” means the procedure described in Example 5and reasonable variations thereof.

“Sugar moiety” means an unmodified sugar moiety or a modified sugarmoiety. As used herein, “unmodified sugar moiety” means a 2′-OH(H)furanosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), ora 2′-H(H) moiety, as found in DNA (an “unmodified DNA sugar moiety”).Unmodified sugar moieties have one hydrogen at each of the 1′, 3′, and4′ positions, an oxygen at the 3′ position, and two hydrogens at the 5′position. As used herein, “modified sugar moiety” or “modified sugar”means a modified furanosyl sugar moiety or a sugar surrogate. As usedherein, modified furanosyl sugar moiety means a furanosyl sugarcomprising a non-hydrogen substituent in place of at least one hydrogenof an unmodified sugar moiety. In certain embodiments, a modifiedfuranosyl sugar moiety is a 2′-substituted sugar moiety. Such modifiedfuranosyl sugar moieties include bicyclic sugars and non-bicyclicsugars. As used herein, “sugar surrogate” means a modified sugar moietyhaving other than a furanosyl moiety that can link a nucleobase toanother group, such as an internucleoside linkage, conjugate group, orterminal group in an oligonucleotide. Modified nucleosides comprisingsugar surrogates can be incorporated into one or more positions withinan oligonucleotide and such oligonucleotides are capable of hybridizingto complementary oligomeric compounds or nucleic acids.

“Target nucleic acid” means a naturally occurring, identified nucleicacid. In certain embodiments, target nucleic acids are endogenouscellular nucleic acids, including, but not limited to RNA transcripts,pre-mRNA, mRNA, microRNA. In certain embodiments, target nucleic acidsare viral nucleic acids. In certain embodiments, target nucleic acidsare nucleic acids that an antisense compound is designed to affect.

“Target region” means a portion of a target nucleic acid to which anantisense compound is designed to hybridize.

“TCA motif” means three nucleosides having the nucleobase sequence TCA(5′-3′). Such nucleosides may have modified sugar moieties and/ormodified internucleosides linkages. Unless otherwise indicated, thenucleosides of TCA motifs comprise unmodified 2′-deoxy sugar moietiesand unmodified phosphodiester internucleoside linkages.

“Terminal group” means a chemical group or group of atoms that iscovalently linked to a terminus of an oligonucleotide.

“CNS” means central nervous system. The CNS includes, the spine and thebrain and the cerebrospinal fluid.

“Cerebrospinal fluid” or “CSF” means the fluid filling the space aroundthe brain and spinal cord.

“Nervous system” means the network of nerve cells and fibers thattransmits nerve impulses between parts of the body. The nervous systemincludes glial cells and neurons. The nervous system includes thecentral nervous system and the peripheral nervous system.

I. Certain Oligonucleotides

In certain embodiments, the invention provides a duplex comprising afirst oligomeric compound and a second oligomeric compound. In certainembodiments an oligomeric compound comprises an oligonucleotide, whichconsists of linked nucleosides. Oligonucleotides may be unmodifiedoligonucleotides (RNA or DNA) or may be modified oligonucleotides.Modified oligonucleotides, for example the first modifiedoligonucleotide or the second modified oligonucleotide, comprise atleast one modification relative to unmodified RNA or DNA (i.e., compriseat least one modified nucleoside (comprising a modified sugar moietyand/or a modified nucleobase) and/or at least one modifiedinternucleoside linkage).

A. Certain Modified Nucleosides

Modified nucleosides comprise a modified sugar moiety or a modifiednucleobase or both a modified sugar moiety and a modified nucleobase.

1. Certain Sugar Moieties

In certain embodiments, modified sugar moieties are non-bicyclicmodified sugar moieties. In certain embodiments, modified sugar moietiesare bicyclic or tricyclic sugar moieties. In certain embodiments,modified sugar moieties are sugar surrogates. Such sugar surrogates maycomprise one or more substitutions corresponding to those of other typesof modified sugar moieties.

In certain embodiments, modified sugar moieties are non-bicyclicmodified sugar moieties comprising a furanosyl ring with one or moreacyclic substituent, including but not limited to substituents at the2′, 4′, and/or 5′ positions. In certain embodiments one or more acyclicsubstituent of non-bicyclic modified sugar moieties is branched.Examples of 2′-substituent groups suitable for non-bicyclic modifiedsugar moieties include but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or“O-methyl”), and 2′-O(CH₂)₂OCH₃ (“MOE”). In certain embodiments,2′-substituent groups are selected from among: halo, allyl, amino,azido, SH, CN, OCN, CF₃, OCF₃, O—C₁-C₁₀ alkoxy, O—C₁-C₁₀ substitutedalkoxy, O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl, S-alkyl,N(R_(m))-alkyl, O-alkenyl, S-alkenyl, N(R_(m))-alkenyl, O-alkynyl,S-alkynyl, N(R_(m))-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl,aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O(CH₂)₂ON(R_(m))(R_(n)) orOCH₂C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is, independently,H, an amino protecting group, or substituted or unsubstituted C₁-C₁₀alkyl, and the 2′-substituent groups described in Cook et al., U.S. Pat.No. 6,531,584; Cook et al., U.S. Pat. No. 5,859,221; and Cook et al.,U.S. Pat. No. 6,005,087. Certain embodiments of these 2′-substituentgroups can be further substituted with one or more substituent groupsindependently selected from among: hydroxyl, amino, alkoxy, carboxy,benzyl, phenyl, nitro (NO₂), thiol, thioalkoxy, thioalkyl, halogen,alkyl, aryl, alkenyl and alkynyl. Examples of 4′-substituent groupssuitable for non-bicyclic modified sugar moieties include but are notlimited to alkoxy (e.g., methoxy), alkyl, and those described inManoharan et al., WO 2015/106128. Examples of 5′-substituent groupssuitable for non-bicyclic modified sugar moieties include but are notlimited to: 5′-methyl (R or S), 5′-vinyl, and 5′-methoxy. In certainembodiments, non-bicyclic modified sugars comprise more than onenon-bridging sugar substituent, for example, 2′-F-5′-methyl sugarmoieties and the modified sugar moieties and modified nucleosidesdescribed in Migawa et al., WO 2008/101157 and Rajeev et al.,US2013/0203836.).

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclicmodified nucleoside comprises a sugar moiety comprising a non-bridging2′-substituent group selected from: F, NH₂, N₃, OCF₃, OCH₃, O(CH₂)₃NH₂,CH₂CH═CH₂, OCH₂CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O(CH₂)₂ON(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (OCH₂C(═O)—N(R_(m))(R_(n))), where each R_(m) and R_(n) is,independently, H, an amino protecting group, or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclicmodified nucleoside comprises a sugar moiety comprising a non-bridging2′-substituent group selected from: F, OCF₃, OCH₃, OCH₂CH₂OCH₃,O(CH₂)₂SCH₃, O(CH₂)₂ON(CH₃)₂, O(CH₂)₂O(CH₂)₂N(CH₃)₂, andOCH₂C(═O)—N(H)CH₃ (“NMA”).

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclicmodified nucleoside comprises a sugar moiety comprising a non-bridging2′-substituent group selected from: F, OCH₃, and OCH₂CH₂OCH₃.

Nucleosides comprising modified sugar moieties, such as non-bicyclicmodified sugar moieties, may be referred to by the position(s) of thesubstitution(s) on the sugar moiety of the nucleoside. For example,nucleosides comprising 2′-substituted or 2-modified sugar moieties arereferred to as 2′-substituted nucleosides or 2-modified nucleosides.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. Incertain such embodiments, the bicyclic sugar moiety comprises a bridgebetween the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′bridging sugar substituents include but are not limited to: 4′-CH₂-2′,4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′ (“LNA”), 4′-CH₂—S-2′,4′-(CH₂)₂—O-2′ (“ENA”), 4′-CH(CH₃)—O-2′ (referred to as “constrainedethyl” or “cEt” when in the S configuration), 4′-CH₂—O—CH₂-2′,4′-CH₂—N(R)-2′, 4′-CH(CH₂OCH₃)—O-2′ (“constrained MOE” or “cMOE”) andanalogs thereof (see, e.g., Seth et al., U.S. Pat. No. 7,399,845, Bhatet al., U.S. Pat. No. 7,569,686, Swayze et al., U.S. Pat. No. 7,741,457,and Swayze et al., U.S. Pat. No. 8,022,193), 4′-C(CH₃)(CH₃)—O-2′ andanalogs thereof (see, e.g., Seth et al., U.S. Pat. No. 8,278,283),4′-CH₂—N(OCH₃)-2′ and analogs thereof (see, e.g., Prakash et al., U.S.Pat. No. 8,278,425), 4′-CH₂—O—N(CH₃)-2′ (see, e.g., Allerson et al.,U.S. Pat. No. 7,696,345 and Allerson et al., U.S. Pat. No. 8,124,745),4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Zhou, et al., J. Org. Chem., 2009, 74,118-134), 4′-CH₂—C(═CH₂)-2′ and analogs thereof (see e.g., Seth et al.,U.S. Pat. No. 8,278,426), 4′-C(R_(a)R_(b))—N(R)—O-2′,4′-C(R_(a)R_(b))—O—N(R)-2′, 4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′,wherein each R, R_(a), and R_(b) is, independently, H, a protectinggroup, or C₁-C₁₂ alkyl (see, e.g. Imanishi et al., U.S. Pat. No.7,427,672).

In certain embodiments, such 4′ to 2′ bridges independently comprisefrom 1 to 4 linked groups independently selected from:—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a))═C(R_(b))—,—C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—,—S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycleradical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl,substituted C₁-C₁₂ aminoalkyl, or a protecting group.

Additional bicyclic sugar moieties are known in the art, see, forexample: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443,Albaek et al., J. Org. Chem., 2006, 71, 7731-7740, Singh et al., Chem.Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54,3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222;Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al.,J. Am. Chem. Soc., 20017, 129, 8362-8379; Wengel et a., U.S. Pat. No.7,053,207; Imanishi et al., U.S. Pat. No. 6,268,490; Imanishi et al.U.S. Pat. No. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al.,U.S. Pat. No. 6,794,499; Wengel et al., U.S. Pat. No. 6,670,461; Wengelet al., U.S. Pat. No. 7,034,133; Wengel et al., U.S. Pat. No. 8,080,644;Wengel et al., U.S. Pat. No. 8,034,909; Wengel et al., U.S. Pat. No.8,153,365; Wengel et al., U.S. Pat. No. 7,572,582; and Ramasamy et al.,U.S. Pat. No. 6,525,191; Torsten et al., WO 2004/106356; Wengel et al.,WO 1999/014226; Seth et al., WO 2007/134181; Seth et al., U.S. Pat. No.7,547,684; Seth et al., U.S. Pat. No. 7,666,854; Seth et al., U.S. Pat.No. 8,088,746; Seth et al., U.S. Pat. No. 7,750,131; Seth et al., U.S.Pat. No. 8,030,467; Seth et al., U.S. Pat. No. 8,268,980; Seth et al.,U.S. Pat. No. 8,546,556; Seth et al., U.S. Pat. No. 8,530,640; Migawa etal., U.S. Pat. No. 9,012,421; Seth et al., U.S. Pat. No. 8,501,805; andU.S. Patent Publication Nos. Allerson et al., US2008/0039618 and Migawaet al., US2015/0191727.

In certain embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, an LNA nucleoside (describedherein) may be in the α-L configuration or in the β-D configuration.

α-L-methyleneoxy (4′-CH₂—O-2′) or α-L-LNA bicyclic nucleosides have beenincorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).Herein, general descriptions of bicyclic nucleosides include bothisomeric configurations. When the positions of specific bicyclicnucleosides (e.g., LNA or cEt) are identified in exemplified embodimentsherein, they are in the β-D configuration, unless otherwise specified.

In certain embodiments, modified sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars).

In certain embodiments, modified sugar moieties are sugar surrogates. Incertain such embodiments, the oxygen atom of the sugar moiety isreplaced, e.g., with a sulfur, carbon or nitrogen atom. In certain suchembodiments, such modified sugar moieties also comprise bridging and/ornon-bridging substituents as described herein. For example, certainsugar surrogates comprise a 4′-sulfur atom and a substitution at the2′-position (see, e.g., Bhat et al., U.S. Pat. No. 7,875,733 and Bhat etal., U.S. Pat. No. 7,939,677) and/or the 5′ position.

In certain embodiments, sugar surrogates comprise rings having otherthan 5 atoms. For example, in certain embodiments, a sugar surrogatecomprises a six-membered tetrahydropyran (“THP”). Such tetrahydropyransmay be further modified or substituted. Nucleosides comprising suchmodified tetrahydropyrans include but are not limited to hexitol nucleicacid (“HNA”), anitol nucleic acid (“ANA”), manitol nucleic acid (“MNA”)(see, e.g., Leumann, C J. Bioorg. & Med. Chem. 2002, 10, 841-854),fluoro HNA:

(“F-HNA”, see e.g. Swayze et al., U.S. Pat. No. 8,088,904; Swayze etal., U.S. Pat. No. 8,440,803; Swayze et al., U.S. Pat. No. 8,796,437;and Swayze et al., U.S. Pat. No. 9,005,906; F-HNA can also be referredto as a F-THP or 3′-fluoro tetrahydropyran), and nucleosides comprisingadditional modified THP compounds having the formula:

wherein, independently, for each of said modified THP nucleoside:

Bx is a nucleobase moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the modified THP nucleoside to the remainder of anoligonucleotide or one of T₃ and T₄ is an internucleoside linking grouplinking the modified THP nucleoside to the remainder of anoligonucleotide and the other of T₃ and T₄ is H, a hydroxyl protectinggroup, a linked conjugate group, or a 5′ or 3′-terminal group; q₁, q₂,q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, or substituted C₂-C₆ alkynyl; and

each of R1 and R2 is independently selected from among: hydrogen,halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ′, and eachJ₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, modified THP nucleosides are provided whereinq₁, q₂, q₃, q₄, q₅, q₆ and are each H. In certain embodiments, at leastone of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other than H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. Incertain embodiments, modified THP nucleosides are provided wherein oneof R₁ and R₂ is F. In certain embodiments, R₁ is F and R₂ is H, incertain embodiments, R₁ is methoxy and R₂ is H, and in certainembodiments, R₁ is methoxyethoxy and R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example, nucleosidescomprising morpholino sugar moieties and their use in oligonucleotideshave been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41,4503-4510 and Summerton et al., U.S. Pat. No. 5,698,685; Summerton etal., U.S. Pat. No. 5,166,315; Summerton et al., U.S. Pat. No. 5,185,444;and Summerton et al., U.S. Pat. No. 5,034,506). As used here, the term“morpholino” means a sugar surrogate having the following structure:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

In certain embodiments, sugar surrogates comprise acyclic moieties.Examples of nucleosides and oligonucleotides comprising such acyclicsugar surrogates include but are not limited to: peptide nucleic acid(“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org.Biomol. Chem., 2013, 11, 5853-5865), and nucleosides andoligonucleotides described in Manoharan et al., WO2011/133876.

Many other bicyclic and tricyclic sugar and sugar surrogate ring systemsare known in the art that can be used in modified nucleosides).

1. Certain Modified Nucleobases

In certain embodiments, the first modified oligonucleotide comprises oneor more nucleoside comprising an unmodified nucleobase. In certainembodiments, the second modified oligonucleotide comprises one or morenucleoside comprising an unmodified nucleobase. In certain embodiments,modified oligonucleotides, for example the first modifiedoligonucleotide or the second modified oligonucleotide, comprise one ormore nucleoside comprising a modified nucleobase. In certainembodiments, modified oligonucleotides, for example the first modifiedoligonucleotide or the second modified oligonucleotide, comprise one ormore nucleoside that does not comprise a nucleobase, referred to as anabasic nucleoside.

In certain embodiments, modified nucleobases are selected from:5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynylsubstituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6substituted purines. In certain embodiments, modified nucleobases areselected from: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine,2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-propynyl (—C≡C—CH₃) uracil, 5-propynylcytosine, 6-azouracil,6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-azaand other 8-substituted purines, 5-halo, particularly 5-bromo,5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine,7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine,7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine,2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases. Further modified nucleobases include tricyclicpyrimidines, such as 1,3-diazaphenoxazine-2-one,1,3-diazaphenothiazine-2-one and9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in Merigan et al., U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859;Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications,Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and thosedisclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T.,Ed., CRC Press, 2008, 163-166 and 442-443.

Publications that teach the preparation of certain of the above notedmodified nucleobases as well as other modified nucleobases includewithout limitation, Manohara et al., US2003/0158403; Manoharan et al.,US2003/0175906; Dinh et al., U.S. Pat. No. 4,845,205; Spielvogel et al.,U.S. Pat. No. 5,130,302; Rogers et al., U.S. Pat. No. 5,134,066;Bischofberger et al., U.S. Pat. No. 5,175,273; Urdea et al., U.S. Pat.No. 5,367,066; Benner et al., U.S. Pat. No. 5,432,272; Matteucci et al.,U.S. Pat. No. 5,434,257; Gmeiner et al., U.S. Pat. No. 5,457,187; Cooket al., U.S. Pat. No. 5,459,255; Froehler et al., U.S. Pat. No.5,484,908; Matteucci et al., U.S. Pat. No. 5,502,177; Hawkins et al.,U.S. Pat. No. 5,525,711; Haralambidis et al., U.S. Pat. No. 5,552,540;Cook et al., U.S. Pat. No. 5,587,469; Froehler et al., U.S. Pat. No.5,594,121; Switzer et al., U.S. Pat. No. 5,596,091; Cook et al., U.S.Pat. No. 5,614,617; Froehler et al., U.S. Pat. No. 5,645,985; Cook etal., U.S. Pat. No. 5,681,941; Cook et al., U.S. Pat. No. 5,811,534; Cooket al., U.S. Pat. No. 5,750,692; Cook et al., U.S. Pat. No. 5,948,903;Cook et al., U.S. Pat. No. 5,587,470; Cook et al., U.S. Pat. No.5,457,191; Matteucci et al., U.S. Pat. No. 5,763,588; Froehler et al.,U.S. Pat. No. 5,830,653; Cook et al., U.S. Pat. No. 5,808,027; Cook etal., U.S. Pat. No. 6,166,199; and Matteucci et al., U.S. Pat. No.6,005,096.

B. Certain Modified Internucleoside Linkages

In certain embodiments, nucleosides of modified oligonucleotides may belinked together using any internucleoside linkage. In certainembodiments, nucleosides of the first modified oligonucleotide may belinked together using any internucleoside linkage. In certainembodiments, nucleosides of the second modified oligonucleotide may belinked together using any internucleoside linkage. The two main classesof internucleoside linking groups are defined by the presence or absenceof a phosphorus atom. Representative phosphorus-containinginternucleoside linkages include but are not limited to phosphates,which contain a phosphodiester bond (“P═O”) (also referred to asunmodified or naturally occurring linkages), phosphotriesters,methylphosphonates, phosphoramidates, and phosphorothioates (“P=5”), andphosphorodithioates (“HS—P═S”). Representative non-phosphorus containinginternucleoside linking groups include but are not limited tomethylenemethylimino (—CH₂—N(CH₃)—O—CH₂—), thiodiester, thionocarbamate(—O—C(═O)(NH)—S—); siloxane (—O—SiH₂—O—); and N,N′-dimethylhydrazine(—CH₂—N(CH₃)—N(CH₃)—). Modified internucleoside linkages, compared tonaturally occurring phosphate linkages, can be used to alter, typicallyincrease, nuclease resistance of the oligonucleotide. In certainembodiments, internucleoside linkages having a chiral atom can beprepared as a racemic mixture, or as separate enantiomers.Representative chiral internucleoside linkages include but are notlimited to alkylphosphonates and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinginternucleoside linkages are well known to those skilled in the art.

Neutral internucleoside linkages include, without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), methoxypropyl, and thioformacetal (3′-S—CH₂—O-5′).Further neutral internucleoside linkages include nonionic linkagescomprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide,sulfide, sulfonate ester and amides (See for example: CarbohydrateModifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds.,ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutralinternucleoside linkages include nonionic linkages comprising mixed N,O, S and CH₂ component parts.

C. Certain Motifs

In certain embodiments, the first modified oligonucleotide comprises oneor more modified nucleoside comprising a modified sugar. In certainembodiments, the first modified oligonucleotide comprises one or moremodified nucleosides comprising a modified nucleobase. In certainembodiments, the first modified oligonucleotide comprises one or moremodified internucleoside linkage. In such embodiments, the modified,unmodified, and differently modified sugar moieties, nucleobases, and/orinternucleoside linkages of the first modified oligonucleotide define apattern or motif. In certain embodiments, the patterns of sugarmoieties, nucleobases, and internucleoside linkages are each independentof one another. Thus, the first modified oligonucleotide may bedescribed by its sugar motif, nucleobase motif and/or internucleosidelinkage motif (as used herein, nucleobase motif describes themodifications to the nucleobases independent of the sequence ofnucleobases).

In certain embodiments, the second modified oligonucleotide comprisesone or more modified nucleoside comprising a modified sugar. In certainembodiments, the second modified oligonucleotide comprises one or moremodified nucleosides comprising a modified nucleobase. In certainembodiments, the second modified oligonucleotide comprises one or moremodified internucleoside linkage. In such embodiments, the modified,unmodified, and differently modified sugar moieties, nucleobases, and/orinternucleoside linkages of the second modified oligonucleotide define apattern or motif. In certain embodiments, the patterns of sugarmoieties, nucleobases, and internucleoside linkages are each independentof one another. Thus, the second modified oligonucleotide may bedescribed by its sugar motif, nucleobase motif and/or internucleosidelinkage motif (as used herein, nucleobase motif describes themodifications to the nucleobases independent of the sequence ofnucleobases).

In certain embodiments, modified oligonucleotides comprise one or moremodified nucleoside comprising a modified sugar. In certain embodiments,modified oligonucleotides comprise one or more modified nucleosidescomprising a modified nucleobase. In certain embodiments, modifiedoligonucleotides comprise one or more modified internucleoside linkage.In such embodiments, the modified, unmodified, and differently modifiedsugar moieties, nucleobases, and/or internucleoside linkages of amodified oligonucleotide define a pattern or motif. In certainembodiments, the patterns of sugar moieties, nucleobases, andinternucleoside linkages are each independent of one another. Thus, amodified oligonucleotide may be described by its sugar motif, nucleobasemotif and/or internucleoside linkage motif (as used herein, nucleobasemotif describes the modifications to the nucleobases independent of thesequence of nucleobases).

1. Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type ofmodified sugar and/or unmodified sugar moiety arranged along theoligonucleotide or region thereof in a defined pattern or sugar motif.In certain instances, such sugar motifs include but are not limited toany of the sugar modifications discussed herein.

In certain embodiments, modified oligonucleotides, for example the firstmodified oligonucleotide, comprise or consist of a region having agapmer motif, which comprises two external regions or “wings” and acentral or internal region or “gap.” The three regions of a gapmer motif(the 5′-wing, the gap, and the 3′-wing) form a contiguous sequence ofnucleosides wherein at least some of the sugar moieties of thenucleosides of each of the wings differ from at least some of the sugarmoieties of the nucleosides of the gap. Specifically, at least the sugarmoieties of the nucleosides of each wing that are closest to the gap(the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside of the3′-wing) differ from the sugar moiety of the neighboring gapnucleosides, thus defining the boundary between the wings and the gap(i.e., the wing/gap junction). In certain embodiments, the sugarmoieties within the gap are the same as one another. In certainembodiments, the gap includes one or more nucleoside having a sugarmoiety that differs from the sugar moiety of one or more othernucleosides of the gap. In certain embodiments, the sugar motifs of thetwo wings are the same as one another (symmetric gapmer). In certainembodiments, the sugar motif of the 5′-wing differs from the sugar motifof the 3′-wing (asymmetric gapmer).

In certain embodiments, the wings of a gapmer comprise 1-5 nucleosides.In certain embodiments, the wings of a gapmer comprise 2-5 nucleosides.In certain embodiments, the wings of a gapmer comprise 3-5 nucleosides.In certain embodiments, the nucleosides of a gapmer are all modifiednucleosides.

In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides.In certain embodiments, the gap of a gapmer comprises 7-10 nucleosides.In certain embodiments, the gap of a gapmer comprises 8-10 nucleosides.In certain embodiments, the gap of a gapmer comprises 10 nucleosides. Incertain embodiment, each nucleoside of the gap of a gapmer is anunmodified 2′-deoxy nucleoside.

In certain embodiments, the gapmer is a deoxy gapmer. In suchembodiments, the nucleosides on the gap side of each wing/gap junctionare unmodified 2′-deoxy nucleosides and the nucleosides on the wingsides of each wing/gap junction are modified nucleosides. In certainsuch embodiments, each nucleoside of the gap is an unmodified 2′-deoxynucleoside. In certain such embodiments, each nucleoside of each wing isa modified nucleoside.

In certain embodiments, the first modified oligonucleotide and/or thesecond modified oligonucleotide comprise or consist of a region having afully modified sugar motif. In such embodiments, each nucleoside of thefully modified region of the modified oligonucleotide comprises amodified sugar moiety. In certain such embodiments, each nucleoside tothe entire modified oligonucleotide (either the first modifiedoligonucleotide and/or the second modified oligonucleotide) comprises amodified sugar moiety. In certain embodiments, modified oligonucleotides(either the first modified oligonucleotide and/or the second modifiedoligonucleotide) comprise or consist of a region having a fully modifiedsugar motif, wherein each nucleoside within the fully modified regioncomprises the same modified sugar moiety, referred to herein as auniformly modified sugar motif. In certain embodiments, a fully modifiedoligonucleotide is a uniformly modified oligonucleotide. In certainembodiments, each nucleoside of a uniformly modified comprises the same2′-modification.

2. Certain Nucleobase Motifs

In certain embodiments, oligonucleotides (including the first modifiedoligonucleotide and/or the second modified oligonucleotide) comprisemodified and/or unmodified nucleobases arranged along theoligonucleotide or region thereof in a defined pattern or motif. Incertain embodiments, each nucleobase is modified. In certainembodiments, none of the nucleobases are modified. In certainembodiments, each purine or each pyrimidine is modified. In certainembodiments, each adenine is modified. In certain embodiments, eachguanine is modified. In certain embodiments, each thymine is modified.In certain embodiments, each uracil is modified. In certain embodiments,each cytosine is modified. In certain embodiments, some or all of thecytosine nucleobases in a modified oligonucleotide are5-methylcytosines.

In certain embodiments, modified oligonucleotides (for example the firstmodified oligonucleotide or the second modified oligonucleotide)comprise a block of modified nucleobases. In certain such embodiments,the block is at the 3′-end of the oligonucleotide. In certainembodiments the block is within 3 nucleosides of the 3′-end of theoligonucleotide. In certain embodiments, the block is at the 5′-end ofthe oligonucleotide. In certain embodiments the block is within 3nucleosides of the 5′-end of the oligonucleotide.

In certain embodiments, oligonucleotides having a gapmer motif comprisea nucleoside comprising a modified nucleobase. In certain suchembodiments, one nucleoside comprising a modified nucleobase is in thecentral gap of an oligonucleotide having a gapmer motif. In certain suchembodiments, the sugar moiety of said nucleoside is a 2′-deoxyribosylmoiety. In certain embodiments, the modified nucleobase is selectedfrom: a 2-thiopyrimidine and a 5-propynepyrimidine. In certainembodiments, the first modified oligonucleotide has a gapmer motif. Incertain embodiments, the first modified oligonucleotide has a gapmermotif and the second modified oligonucleotide does not have a gapmermotif. In certain embodiments, the first modified oligonucleotide has agapmer motif and the second modified oligonucleotide has a fullymodified motif

3. Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides, for example the first modifiedoligonucleotide and/or the second modified oligonucleotide, comprisemodified and/or unmodified internucleoside linkages arranged along theoligonucleotide or region thereof in a defined pattern or motif. Incertain embodiments, essentially each internucleoside linking group is aphosphate internucleoside linkage (P═O). In certain embodiments, eachinternucleoside linking group of a modified oligonucleotide is aphosphorothioate (P═S). In certain embodiments, essentially eachinternucleoside linking group of the first modified oligonucleotide is aphosphate internucleoside linkage (P═O). In certain embodiments, eachinternucleoside linking group of the first modified oligonucleotide is aphosphorothioate (P═S). In certain embodiments, essentially eachinternucleoside linking group of the second modified oligonucleotide isa phosphate internucleoside linkage (P═O). In certain embodiments, eachinternucleoside linking group of the second modified oligonucleotide isa phosphorothioate (P═S). In certain embodiments, each internucleosidelinking group of the first modified oligonucleotide is independentlyselected from a phosphorothioate and phosphate internucleoside linkage.In certain embodiments, each internucleoside linking group of the secondmodified oligonucleotide is independently selected from aphosphorothioate and phosphate internucleoside linkage. In certainembodiments, the sugar motif of the first modified oligonucleotide is agapmer and the internucleoside linkages within the gap are all modified.In certain such embodiments, some or all of the internucleoside linkagesin the wings are unmodified phosphate linkages. In certain embodiments,the terminal internucleoside linkages are modified.

D. Certain Lengths

In certain embodiments, oligonucleotides (including modifiedoligonucleotides) can have any of a variety of ranges of lengths. Incertain embodiments, the first modified oligonucleotide can have any ofa variety of ranges of lengths. In certain embodiments, the secondmodified oligonucleotide can have any of a variety of ranges of lengths.In certain embodiments, oligonucleotides consist of X to Y linkednucleosides, where X represents the fewest number of nucleosides in therange and Y represents the largest number nucleosides in the range. Incertain such embodiments, X and Y are each independently selected from8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, and 50; provided that X≤Y. For example, incertain embodiments, oligonucleotides consist of 12 to 13, 12 to 14, 12to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to30 linked nucleosides

E. Certain Modified Oligonucleotides

In certain embodiments, the above modifications (sugar, nucleobase,internucleoside linkage) are incorporated into a modifiedoligonucleotide. In certain embodiments, modified oligonucleotides arecharacterized by their modification motifs and overall lengths. Incertain embodiments, such parameters are each independent of oneanother. Thus, unless otherwise indicated, each internucleoside linkageof an oligonucleotide having a gapmer sugar motif may be modified orunmodified and may or may not follow the gapmer modification pattern ofthe sugar modifications. For example, the internucleoside linkageswithin the wing regions of a sugar gapmer may be the same or differentfrom one another and may be the same or different from theinternucleoside linkages of the gap region of the sugar motif. Likewise,such sugar gapmer oligonucleotides may comprise one or more modifiednucleobase independent of the gapmer pattern of the sugar modifications.Furthermore, in certain instances, an oligonucleotide is described by anoverall length or range and by lengths or length ranges of two or moreregions (e.g., a regions of nucleosides having specified sugarmodifications), in such circumstances it may be possible to selectnumbers for each range that result in an oligonucleotide having anoverall length falling outside the specified range. In suchcircumstances, both elements must be satisfied. For example, in certainembodiments, a modified oligonucleotide consists if of 15-20 linkednucleosides and has a sugar motif consisting of three regions, A, B, andC, wherein region A consists of 2-6 linked nucleosides having aspecified sugar motif, region B consists of 6-10 linked nucleosideshaving a specified sugar motif, and region C consists of 2-6 linkednucleosides having a specified sugar motif. Such embodiments do notinclude modified oligonucleotides where A and C each consist of 6 linkednucleosides and B consists of 10 linked nucleosides (even though thosenumbers of nucleosides are permitted within the requirements for A, B,and C) because the overall length of such oligonucleotide is 22, whichexceeds the upper limit of the overall length of the modifiedoligonucleotide (20). Herein, if a description of an oligonucleotide issilent with respect to one or more parameter, such parameter is notlimited. Thus, a modified oligonucleotide described only as having agapmer sugar motif without further description may have any length,internucleoside linkage motif, and nucleobase motif. Unless otherwiseindicated, all modifications are independent of nucleobase sequence.

F. Nucleobase Sequence

In certain embodiments, oligonucleotides (unmodified or modifiedoligonucleotides) are further described by their nucleobase sequence. Incertain embodiments oligonucleotides have a nucleobase sequence that iscomplementary to a second oligonucleotide or an identified referencenucleic acid, such as a target nucleic acid. In certain suchembodiments, a region of an oligonucleotide has a nucleobase sequencethat is complementary to a second oligonucleotide or an identifiedreference nucleic acid, such as a target nucleic acid. In certainembodiments, the nucleobase sequence of a region or entire length of anoligonucleotide is at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or 100% complementary to the secondoligonucleotide or nucleic acid, such as a target nucleic acid.

II. Certain Oligomeric Compounds

In certain embodiments, the invention provides oligomeric compounds,which consist of an oligonucleotide (modified or unmodified or a firstmodified oligonucleotide or a second modified oligonucleotide) andoptionally one or more conjugate groups and/or terminal groups.Conjugate groups consist of one or more conjugate moiety and a conjugatelinker which links the conjugate moiety to the oligonucleotide.Conjugate groups may be attached to either or both ends of anoligonucleotide and/or at any internal position. In certain embodiments,conjugate groups are attached to the 2′-position of a nucleoside of amodified oligonucleotide. In certain embodiments, conjugate groups thatare attached to either or both ends of an oligonucleotide are terminalgroups. In certain such embodiments, conjugate groups or terminal groupsare attached at the 3′ and/or 5′-end of oligonucleotides. In certainsuch embodiments, conjugate groups (or terminal groups) are attached atthe 3′-end of oligonucleotides. In certain embodiments, conjugate groupsare attached near the 3′-end of oligonucleotides. In certainembodiments, conjugate groups (or terminal groups) are attached at the5′-end of oligonucleotides. In certain embodiments, conjugate groups areattached near the 5′-end of oligonucleotides.

Examples of terminal groups include but are not limited to conjugategroups, capping groups, phosphate moieties, protecting groups, modifiedor unmodified nucleosides, and two or more nucleosides that areindependently modified or unmodified.

A. Certain Conjugate Groups

In certain embodiments, oligonucleotides, including a first modifiedoligonucleotide or a second modified oligonucleotide, are covalentlyattached to one or more conjugate groups. In certain embodiments, asecond modified oligonucleotide is covalently attached to one or moreconjugate groups. In certain embodiments, a second modifiedoligonucleotide is covalently attached to one or more conjugate groupsand the first modified oligonucleotide is not attached to a conjugategroup. In certain embodiments, conjugate groups modify one or moreproperties of the attached oligonucleotide, including but not limited topharmacodynamics, pharmacokinetics, stability, binding, absorption,tissue distribution, cellular distribution, cellular uptake, charge andclearance. In certain embodiments, conjugate groups impart a newproperty on the attached oligonucleotide, e.g., fluorophores or reportergroups that enable detection of the oligonucleotide. Certain conjugategroups and conjugate moieties have been described previously, forexample: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med.Chem. Lett., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937), a tocopherol group (Nishina et al.,Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al.,Molecular Therapy, 2008, 16, 734-740), or a GalNAc cluster (e.g.,WO2014/179620).

1. Conjugate Moieties

Conjugate moieties include, without limitation, intercalators, reportermolecules, polyamines, polyamides, peptides, carbohydrates, vitaminmoieties, polyethylene glycols, thioethers, polyethers, cholesterols,thiocholesterols, cholic acid moieties, folate, lipids, phospholipids,biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine,fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

In certain embodiments, a conjugate moiety comprises an active drugsubstance, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic.

2. Conjugate Linkers

Conjugate moieties are attached to oligonucleotides through conjugatelinkers. In certain oligomeric compounds, the conjugate linker is asingle chemical bond (i.e., the conjugate moiety is attached directly toan oligonucleotide through a single bond). In certain embodiments, theconjugate linker comprises a chain structure, such as a hydrocarbylchain, or an oligomer of repeating units such as ethylene glycol,nucleosides, or amino acid units.

In certain embodiments, a conjugate linker comprises one or more groupsselected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol,ether, thioether, and hydroxylamino. In certain such embodiments, theconjugate linker comprises groups selected from alkyl, amino, oxo, amideand ether groups. In certain embodiments, the conjugate linker comprisesgroups selected from alkyl and amide groups. In certain embodiments, theconjugate linker comprises groups selected from alkyl and ether groups.In certain embodiments, the conjugate linker comprises at least onephosphorus moiety. In certain embodiments, the conjugate linkercomprises at least one phosphate group. In certain embodiments, theconjugate linker includes at least one neutral linking group.

In certain embodiments, conjugate linkers, including the conjugatelinkers described above, are bifunctional linking moieties, e.g., thoseknown in the art to be useful for attaching conjugate groups to parentcompounds, such as the oligonucleotides provided herein. In general, abifunctional linking moiety comprises at least two functional groups.One of the functional groups is selected to bind to a particular site ona parent compound and the other is selected to bind to a conjugategroup. Examples of functional groups used in a bifunctional linkingmoiety include but are not limited to electrophiles for reacting withnucleophilic groups and nucleophiles for reacting with electrophilicgroups. In certain embodiments, bifunctional linking moieties compriseone or more groups selected from amino, hydroxyl, carboxylic acid,thiol, alkyl, alkenyl, and alkynyl.

Examples of conjugate linkers include but are not limited topyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include butare not limited to substituted or unsubstituted C₁-C₁₀ alkyl,substituted or unsubstituted C₂-C₁₀ alkenyl or substituted orunsubstituted C₂-C₁₀ alkynyl, wherein a nonlimiting list of preferredsubstituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl.

In certain embodiments, conjugate linkers comprise 1-10linker-nucleosides. In certain embodiments, conjugate linkers comprise2-5 linker-nucleosides. In certain embodiments, conjugate linkerscomprise exactly 3 linker-nucleosides. In certain embodiments, conjugatelinkers comprise the TCA motif. In certain embodiments, suchlinker-nucleosides are modified nucleosides. In certain embodiments suchlinker-nucleosides comprise a modified sugar moiety. In certainembodiments, linker-nucleosides are unmodified. In certain embodiments,linker-nucleosides comprise an optionally protected heterocyclic baseselected from a purine, substituted purine, pyrimidine or substitutedpyrimidine. In certain embodiments, a cleavable moiety is a nucleosideselected from uracil, thymine, cytosine, 4-N-benzoylcytosine,5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine,6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typicallydesirable for linker-nucleosides to be cleaved from the oligomericcompound after it reaches a target tissue. Accordingly,linker-nucleosides are typically linked to one another and to theremainder of the oligomeric compound through cleavable bonds. In certainembodiments, such cleavable bonds are phosphodiester bonds.

Herein, linker-nucleosides are not considered to be part of theoligonucleotide. Accordingly, in embodiments in which an oligomericcompound comprises an oligonucleotide consisting of a specified numberor range of linked nucleosides and/or a specified percentcomplementarity to a reference nucleic acid and the oligomeric compoundalso comprises a conjugate group comprising a conjugate linkercomprising linker-nucleosides, those linker-nucleosides are not countedtoward the length of the oligonucleotide and are not used in determiningthe percent complementarity of the oligonucleotide for the referencenucleic acid. For example, an oligomeric compound may comprise (1) amodified oligonucleotide consisting of 8-30 nucleosides and (2) aconjugate group comprising 1-10 linker-nucleosides that are contiguouswith the nucleosides of the modified oligonucleotide. The total numberof contiguous linked nucleosides in such an oligomeric compound is morethan 30. Alternatively, an oligomeric compound may comprise a modifiedoligonucleotide consisting of 8-30 nucleosides and no conjugate group.The total number of contiguous linked nucleosides in such an oligomericcompound is no more than 30. Unless otherwise indicated conjugatelinkers comprise no more than 10 linker-nucleosides. In certainembodiments, conjugate linkers comprise no more than 5linker-nucleosides. In certain embodiments, conjugate linkers compriseno more than 3 linker-nucleosides. In certain embodiments, conjugatelinkers comprise no more than 2 linker-nucleosides. In certainembodiments, conjugate linkers comprise no more than 1linker-nucleoside.

In certain embodiments, it is desirable for a conjugate group to becleaved from the oligonucleotide. For example, in certain circumstancesoligomeric compounds comprising a particular conjugate moiety are bettertaken up by a particular cell type, but once the oligomeric compound hasbeen taken up, it is desirable that the conjugate group be cleaved torelease the unconjugated or parent oligonucleotide. Thus, certainconjugate linkers may comprise one or more cleavable moieties. Incertain embodiments, a cleavable moiety is a cleavable bond. In certainembodiments, a cleavable moiety is a group of atoms comprising at leastone cleavable bond. In certain embodiments, a cleavable moiety comprisesa group of atoms having one, two, three, four, or more than fourcleavable bonds. In certain embodiments, a cleavable moiety isselectively cleaved inside a cell or subcellular compartment, such as alysosome. In certain embodiments, a cleavable moiety is selectivelycleaved by endogenous enzymes, such as nucleases.

In certain embodiments, a cleavable bond is selected from among: anamide, an ester, an ether, one or both esters of a phosphodiester, aphosphate ester, a carbamate, or a disulfide. In certain embodiments, acleavable bond is one or both of the esters of a phosphodiester. Incertain embodiments, a cleavable moiety comprises a phosphate orphosphodiester. In certain embodiments, the cleavable moiety is aphosphate linkage between an oligonucleotide and a conjugate moiety orconjugate group.

In certain embodiments, a cleavable moiety comprises or consists of oneor more linker-nucleosides. In certain such embodiments, the one or morelinker-nucleosides are linked to one another and/or to the remainder ofthe oligomeric compound through cleavable bonds. In certain embodiments,such cleavable bonds are unmodified phosphodiester bonds. In certainembodiments, a cleavable moiety is 2′-deoxy nucleoside that is attachedto either the 3′ or 5′-terminal nucleoside of an oligonucleotide by aphosphate internucleoside linkage and covalently attached to theremainder of the conjugate linker or conjugate moiety by a phosphate orphosphorothioate linkage. In certain such embodiments, the cleavablemoiety is 2′-deoxyadenosine.

III. Certain Antisense Compounds

In certain embodiments, the present invention provides antisensecompounds, which comprise or consist of an oligomeric compoundcomprising an antisense oliognucleotide, having a nucleobase sequencescomplementary to that of a target nucleic acid. In certain embodiments,antisense compounds are single-stranded. Such single-stranded antisensecompounds typically comprise or consist of an oligomeric compound thatcomprises or consists of a modified oligonucleotide and optionally aconjugate group. In certain embodiments, antisense compounds aredouble-stranded. Such double-stranded antisense compounds comprise afirst oligomeric compound having a region complementary to a targetnucleic acid and a second oligomeric compound having a regioncomplementary to the first oligomeric compound. The first oligomericcompound of such double stranded antisense compounds typically comprisesor consists of a modified oligonucleotide and optionally a conjugategroup. The oligonucleotide of the second oligomeric compound of suchdouble-stranded antisense compound may be modified or unmodified. Eitheror both oligomeric compounds of a double-stranded antisense compound maycomprise a conjugate group. The oligomeric compounds of double-strandedantisense compounds may include non-complementary overhangingnucleosides.

In certain embodiments, oligomeric compounds of antisense compounds arecapable of hybridizing to a target nucleic acid, resulting in at leastone antisense activity. In certain embodiments, antisense compoundsselectively affect one or more target nucleic acid. Such selectiveantisense compounds comprises a nucleobase sequence that hybridizes toone or more target nucleic acid, resulting in one or more desiredantisense activity and does not hybridize to one or more non-targetnucleic acid or does not hybridize to one or more non-target nucleicacid in such a way that results in significant undesired antisenseactivity.

In certain antisense activities, hybridization of an antisense compoundto a target nucleic acid results in recruitment of a protein thatcleaves the target nucleic acid. For example, certain antisensecompounds result in RNase H mediated cleavage of the target nucleicacid. RNase H is a cellular endonuclease that cleaves the RNA strand ofan RNA:DNA duplex. The DNA in such an RNA:DNA duplex need not beunmodified DNA. In certain embodiments, the invention provides antisensecompounds that are sufficiently “DNA-like” to elicit RNase H activity.Further, in certain embodiments, one or more non-DNA-like nucleoside inthe gap of a gapmer is tolerated.

In certain antisense activities, an antisense compound or a portion ofan antisense compound is loaded into an RNA-induced silencing complex(RISC), ultimately resulting in cleavage of the target nucleic acid. Forexample, certain antisense compounds result in cleavage of the targetnucleic acid by Argonaute. Antisense compounds that are loaded into RISCare RNAi compounds. RNAi compounds may be double-stranded (siRNA) orsingle-stranded (ssRNA).

In certain embodiments, hybridization of an antisense compound to atarget nucleic acid does not result in recruitment of a protein thatcleaves that target nucleic acid. In certain such embodiments,hybridization of the antisense compound to the target nucleic acidresults in alteration of splicing of the target nucleic acid. In certainembodiments, hybridization of an antisense compound to a target nucleicacid results in inhibition of a binding interaction between the targetnucleic acid and a protein or other nucleic acid. In certain suchembodiments, hybridization of an antisense compound to a target nucleicacid results in alteration of translation of the target nucleic acid.

Antisense activities may be observed directly or indirectly. In certainembodiments, observation or detection of an antisense activity involvesobservation or detection of a change in an amount of a target nucleicacid or protein encoded by such target nucleic acid, a change in theratio of splice variants of a nucleic acid or protein, and/or aphenotypic change in a cell or animal.

IV. Certain Duplexes

In certain embodiments, the present invention provides duplexescomprising a first oligomeric compound comprising a first modifiedoligonucleotide and a second oligomeric compound comprising a secondoligonucleotide, wherein the first and second modified oligonucleotidescomprise regions of complementarity sufficient to form a duplex. Incertain such embodiments, the first modified oligonucleotide iscomplementary to a target nucleic acid. In certain such embodiments, thefirst modified oligonucleotide is a gapmer as described above. Thus, insuch embodiments, the first oligomeric compound is capable ofhybridizing to a target nucleic acid and eliciting cleavage of thetarget nucleic acid by RNase H. In certain such embodiments, the secondoligomeric compound improves a property of the first oligomeric compoundcompared to the property in the absence of the second oligomericcompound. In certain embodiments, that improved property is one or moreof: distribution to a target tissue, uptake into a target cell, potency,and efficacy. In certain embodiments, the target tissue is liver(hepatic). In certain embodiments, the target tissue is other than liver(extra-hepatic). In certain embodiments, it is desirable to reducetarget in more than one tissue. In certain such embodiments, it isdesirable to reduce target in the liver and one or more other tissues.In certain embodiments, it is desirable to reduce target in more thanone extra-hepatic tissue.

In certain embodiments, the first oligonucleotide of a duplex is agapmer. In certain such embodiments, the wings of the gapmer comprise2′-MOE modified nucleosides. In certain embodiments, the wings of thegapmer comprise cEt nucleosides. In certain embodiments the wings of thegapmer comprise LNA nucleosides. In certain embodiments, the wings of agapmer comprise at least one 2′-MOE modified nucleoside and at least onebicyclic nucleoside. In certain such embodiments, each such bicyclicnucleoside is selected from among an LNA nucleoside and a cEtnucleoside. In certain embodiments, the gap constitutes 7-102′-deoxynucleosides.

In certain embodiments, the second oligonucleotide comprises at leastone 2′-MOE nucleoside. In certain embodiments, the secondoligonucleotide comprises 2′-MOE and 2′-deoxynucleosides. In certainembodiments, the second oligonucleotide has sugar motif of alternatingmodification types (including no modification). In certain suchembodiments, the sugar motif of the second oligonucleotide alternatesbetween 2′-MOE nucleosides and 2′-deoxynucleosides. In certainembodiments, the second oligonucleotide has a sugar motif similar to agapmer (as described above) except that it may not elicit cleavage of atarget nucleic acid. Such gapmer-like motifs have a central region andflanking wing regions. In certain such embodiment, the central region iscomprises of 2′-deoxynucleosides and the wing regions are 2′-MOEmodified nucleosides. The internucleoside linkages of the secondoligonucleotide may be modified or phosphodiester. In certainembodiments, the internucleoside linkages of the second oligonucleotidefollow a gapmer-like motif—phosphorothioate wings and phosphdiester inthe center. Such internucleoside linkage motif may or may not track thesugar motif. Though duplexes comprising second oligomeric compounds withsecond oligonucleotides having central regions comprising RNA are shownherein to have enhanced activity (compared to the first oligomericcompound alone and not in a duplex) it is noted that sucholigonucleotides comprising RNA are expensive to manufacture andrelatively unstable when compared to oligonucleotides that comprisemodified nucleosides or DNA nucleosides.

In certain embodiments, at least one of the first and second oligomericcompounds comprises a conjugate group (as described above). Typically,the second oligomeric compound comprises a conjugate group. Theconjugate group may be attached at either end of the oligomericcompound. In certain embodiments, a conjugate group is attached to bothends.

V. Certain Target Nucleic Acids

In certain embodiments, antisense compounds comprise or consist of anoligonucleotide comprising a region that is complementary to a targetnucleic acid. In certain embodiments, the target nucleic acid is anendogenous RNA molecule. In certain embodiments, the target nucleic acidencodes a protein. In certain such embodiments, the target nucleic acidis selected from: an mRNA and a pre-mRNA, including intronic, exonic anduntranslated regions. In certain embodiments, the target RNA is an mRNA.In certain embodiments, the target nucleic acid is a pre-mRNA. Incertain such embodiments, the target region is entirely within anintron. In certain embodiments, the target region spans an intron/exonjunction. In certain embodiments, the target region is at least 50%within an intron.

In certain embodiments, the target nucleic acid is a non-coding RNA. Incertain such embodiments, the target non-coding RNA is selected from: along-non-coding RNA, a short non-coding RNA, an intronic RNA molecule, asnoRNA, a scaRNA, a microRNA (including pre-microRNA and maturemicroRNA), a ribosomal RNA, and promoter directed RNA. In certainembodiments, the target nucleic acid is a nucleic acid other than amature mRNA. In certain embodiments, the target nucleic acid is anucleic acid other than a mature mRNA or a microRNA. In certainembodiments, the target nucleic acid is a non-coding RNA other than amicroRNA. In certain embodiments, the target nucleic acid is anon-coding RNA other than a microRNA or an intronic region of apre-mRNA. In certain embodiments, the target nucleic acid is a longnon-coding RNA. In certain embodiments, the target nucleic acid is anon-coding RNA associated with splicing of other pre-mRNAs. In certainembodiments, the target nucleic acid is a nuclear-retained non-codingRNA.

In certain embodiments, antisense compounds described herein arecomplementary to a target nucleic acid comprising a single-nucleotidepolymorphism (SNP). In certain such embodiments, the antisense compoundis capable of modulating expression of one allele of the SNP-containingtarget nucleic acid to a greater or lesser extent than it modulatesanother allele. In certain embodiments, an antisense compound hybridizesto a (SNP)-containing target nucleic acid at the single-nucleotidepolymorphism site.

In certain embodiments, antisense compounds are at least partiallycomplementary to more than one target nucleic acid. For example,antisense compounds of the present invention may mimic microRNAs, whichtypically bind to multiple targets.

A. Complementarity/Mismatches to the Target Nucleic Acid

In certain embodiments, antisense compounds comprise antisenseoligonucleotides that are complementary to the target nucleic acid overthe entire length of the oligonucleotide. In certain embodiments, sucholigonucleotides are 99% complementary to the target nucleic acid. Incertain embodiments, such oligonucleotides are 95% complementary to thetarget nucleic acid. In certain embodiments, such oligonucleotides are90% complementary to the target nucleic acid. In certain embodiments,such oligonucleotides are 85% complementary to the target nucleic acid.In certain embodiments, such oligonucleotides are 80% complementary tothe target nucleic acid. In certain embodiments, antisenseoligonucleotides are at least 80% complementary to the target nucleicacid over the entire length of the oligonucleotide and comprise a regionthat is 100% or fully complementary to a target nucleic acid. In certainsuch embodiments, the region of full complementarity is from 6 to 20nucleobases in length. In certain such embodiments, the region of fullcomplementarity is from 10 to 18 nucleobases in length. In certain suchembodiments, the region of full complementarity is from 18 to 20nucleobases in length.

In certain embodiments, the oligomeric compounds of antisense compoundscomprise one or more mismatched nucleobases relative to the targetnucleic acid. In certain such embodiments, antisense activity againstthe target is reduced by such mismatch, but activity against anon-target is reduced by a greater amount. Thus, in certain suchembodiments selectivity of the antisense compound is improved. Incertain embodiments, the mismatch is specifically positioned within anoligonucleotide having a gapmer motif. In certain such embodiments, themismatch is at position 1, 2, 3, 4, 5, 6, 7, or 8 from the 5′-end of thegap region. In certain such embodiments, the mismatch is at position 9,8, 7, 6, 5, 4, 3, 2, 1 from the 3′-end of the gap region. In certainsuch embodiments, the mismatch is at position 1, 2, 3, or 4 from the5′-end of the wing region. In certain such embodiments, the mismatch isat position 4, 3, 2, or 1 from the 3′-end of the wing region.

B. Certain Target Nucleic Acids in Certain Tissues

In certain embodiments, antisense compounds comprise or consist of anoligonucleotide comprising a region that is complementary to a targetnucleic acid, wherein the target nucleic acid is expressed in anextra-hepatic tissue. Extra-hepatic tissues include, but are not limitedto: skeletal muscle, cardiac muscle, smooth muscle, adipose, whiteadipose, spleen, bone, intestine, adrenal, testes, ovary, pancreas,pituitary, prostate, skin, uterus, bladder, brain, glomerulus, distaltubular epithelium, breast, lung, heart, kidney, ganglion, frontalcortex, spinal cord, trigeminal ganglia, sciatic nerve, dorsal rootganglion, epididymal fat, diaphragm, pancreas, and colon.

A. Certain First Modified Oligonucleotides

In certain embodiments, disclosed here in are first modifiedoligonucleotides designed to target certain nucleic acid targets. TablesA and B below describe certain modified oligonucleotides targeted tocertain nucleic acid transcripts. In Tables A and B below, subscript “s”represents a phosphorothioate internucleoside linkage, subscript “o”represents a phosphate internucleoside linkage, subscript “d” representsa 2′-deoxynucleoside, subscript “e” represents a 2′-MOE modifiednucleoside, and subscript “k” represents a cEt modified nucleoside. Intables A and B below, superscript “m” before a C represents a5-methylcysteine.

TABLE A Certain First Modified Oligonucleotides SEQ Isis ID Target No.Sequence (5′-3′) Motif NO: CRP 329993 AGCATAGTTAACGAGCTCCC 5-10-5 MOE 14PTPB1B 404173 AATGGTTTATTCCATGGCCA 5-10-5 MOE 15 GCCR 426115GCAGCCATGGTGATCAGGAG 5-10-5 MOE 16 GCGR 449884 GGTTCCCGAGGTGCCCA3-10-4 MOE 17 FGFR4 463588 GCACACTCAGCAGGACCCCC 5-10-5 MOE 18 GHr 532401CCACCTTTGGGTGAATAGCA 5-10-5 MOE 19 DGAT2 484137 TGCCATTTAATGAGCTTCAC5-10-5 MOE 20 DMPK 598769 TCCCGAATGTCCGACA Mixed wing 21 CFB 696844ATCCCACGCCCCTGTCCAGC 5-10-5 MOE 22 GalNAc

TABLE B Certain First Modified Oligonucleotides Target Isis No.Motif (5′-3′) CRP 329993 A_(es)G_(es)^(m)C_(es)A_(es)T_(es)A_(ds)G_(ds)T_(ds)T_(ds)A_(ds)A_(ds)^(m)C_(ds)G_(ds)A_(ds)G_(ds) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(e) PTPB1B 404173A_(es)A_(es)T_(es)G_(es)G_(es)T_(ds)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(es)G_(es) ^(m)C_(es) ^(m)C_(es)A_(e)GCCR 426115 G_(es) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)^(m)C_(ds)A_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(as)A_(ds)T_(ds)^(m)C_(ds)A_(es)G_(es)G_(es)A_(es)G_(e) GCGR 449884G_(es)G_(es)T_(es)T_(ds) ^(m)C_(ds) ^(m)C_(ds)^(m)C_(ds)G_(ds)A_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(e) FGFR4 463588 G_(es) ^(m)C_(es)A_(es) ^(m)C_(es)A_(es)^(m)C_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)A_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) GHr 532401 ^(m)C_(es) ^(m)C_(es)A_(es) ^(m)C_(es)^(m)C_(es)T_(ds)T_(ds)T_(ds)G_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)A_(ds)T_(es)A_(es)G_(es)^(m)C_(es)A_(e) DGAT2 484137 T_(es)G_(es) ^(m)C_(es)^(m)C_(es)A_(es)T_(ds)T_(ds)T_(ds)A_(ds)A_(ds)T_(ds)G_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es) ^(m)C_(es)A_(es) ^(m)C_(e) DMPK 598769 T_(es)^(m)C_(es) ^(m)C_(ks) ^(m)C_(ks)G_(ds)A_(ds)A_(ds)T_(ds)G_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds)G_(ks)A_(ks) ^(m)C_(es)A_(e) CFB 696844A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(ds) ^(m)C_(ds)G_(ds)^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(es)^(m)C_(es)A_(es)G_(es) ^(m)C_(e)The first modified oligonucleotides provided above can be paired withthe second modified oligonucleotide of a second oligomeric compound toform a duplex.

I. Certain Pharmaceutical Compositions

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising one or more antisense compound or a saltthereof. In certain such embodiments, the pharmaceutical compositioncomprises a suitable pharmaceutically acceptable diluent or carrier. Incertain embodiments, a pharmaceutical composition comprises a sterilesaline solution and one or more antisense compound. In certainembodiments, such pharmaceutical composition consists of a sterilesaline solution and one or more antisense compound. In certainembodiments, the sterile saline is pharmaceutical grade saline. Incertain embodiments, a pharmaceutical composition comprises one or moreantisense compound and sterile water. In certain embodiments, apharmaceutical composition consists of one antisense compound andsterile water. In certain embodiments, the sterile water ispharmaceutical grade water. In certain embodiments, a pharmaceuticalcomposition comprises one or more antisense compound andphosphate-buffered saline (PBS). In certain embodiments, apharmaceutical composition consists of one or more antisense compoundand sterile PBS. In certain embodiments, the sterile PBS ispharmaceutical grade PBS.

In certain embodiments, pharmaceutical compositions comprise one or moreor antisense compound and one or more excipients. In certain suchembodiments, excipients are selected from water, salt solutions,alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesiumstearate, talc, silicic acid, viscous paraffin, hydroxymethylcelluloseand polyvinylpyrrolidone.

In certain embodiments, antisense compounds may be admixed withpharmaceutically acceptable active and/or inert substances for thepreparation of pharmaceutical compositions or formulations. Compositionsand methods for the formulation of pharmaceutical compositions depend ona number of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

In certain embodiments, pharmaceutical compositions comprising anantisense compound encompass any pharmaceutically acceptable salts ofthe antisense compound, esters of the antisense compound, or salts ofsuch esters. In certain embodiments, pharmaceutical compositionscomprising antisense compounds comprising one or more antisenseoligonucleotide, upon administration to an animal, including a human,are capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to pharmaceutically acceptable salts ofantisense compounds, prodrugs, pharmaceutically acceptable salts of suchprodrugs, and other bioequivalents. Suitable pharmaceutically acceptablesalts include, but are not limited to, sodium and potassium salts. Incertain embodiments, prodrugs comprise one or more conjugate groupattached to an oligonucleotide, wherein the conjugate group is cleavedby endogenous nucleases within the body.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In certain such methods, the nucleic acid, such as an antisensecompound, is introduced into preformed liposomes or lipoplexes made ofmixtures of cationic lipids and neutral lipids. In certain methods, DNAcomplexes with mono- or poly-cationic lipids are formed without thepresence of a neutral lipid. In certain embodiments, a lipid moiety isselected to increase distribution of a pharmaceutical agent to aparticular cell or tissue. In certain embodiments, a lipid moiety isselected to increase distribution of a pharmaceutical agent to fattissue. In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions comprise a deliverysystem. Examples of delivery systems include, but are not limited to,liposomes and emulsions. Certain delivery systems are useful forpreparing certain pharmaceutical compositions including those comprisinghydrophobic compounds. In certain embodiments, certain organic solventssuch as dimethylsulfoxide are used.

In certain embodiments, pharmaceutical compositions comprise one or moretissue-specific delivery molecules designed to deliver the one or morepharmaceutical agents of the present invention to specific tissues orcell types. For example, in certain embodiments, pharmaceuticalcompositions include liposomes coated with a tissue-specific antibody.

In certain embodiments, pharmaceutical compositions comprise aco-solvent system. Certain of such co-solvent systems comprise, forexample, benzyl alcohol, a nonpolar surfactant, a water-miscible organicpolymer, and an aqueous phase. In certain embodiments, such co-solventsystems are used for hydrophobic compounds. A non-limiting example ofsuch a co-solvent system is the VPD co-solvent system, which is asolution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v ofthe nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol300. The proportions of such co-solvent systems may be variedconsiderably without significantly altering their solubility andtoxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, pharmaceutical compositions are prepared fororal administration. In certain embodiments, pharmaceutical compositionsare prepared for buccal administration. In certain embodiments, apharmaceutical composition is prepared for administration by injection(e.g., intravenous, subcutaneous, intramuscular, etc.). In certain ofsuch embodiments, a pharmaceutical composition comprises a carrier andis formulated in aqueous solution, such as water or physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer. In certain embodiments, other ingredientsare included (e.g., ingredients that aid in solubility or serve aspreservatives). In certain embodiments, injectable suspensions areprepared using appropriate liquid carriers, suspending agents and thelike. Certain pharmaceutical compositions for injection are presented inunit dosage form, e.g., in ampoules or in multi-dose containers. Certainpharmaceutical compositions for injection are suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents. Certainsolvents suitable for use in pharmaceutical compositions for injectioninclude, but are not limited to, lipophilic solvents and fatty oils,such as sesame oil, synthetic fatty acid esters, such as ethyl oleate ortriglycerides, and liposomes. Aqueous injection suspensions may contain.

Nonlimiting Disclosure and Incorporation by Reference

Each of the literature and patent publications listed herein isincorporated by reference in its entirety. While certain compounds,compositions and methods described herein have been described withspecificity in accordance with certain embodiments, the followingexamples serve only to illustrate the compounds described herein and arenot intended to limit the same. Each of the references, GenBankaccession numbers, and the like recited in the present application isincorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar (2′-OH in placeof one 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) in place of a uracil of RNA). Accordingly, nucleicacid sequences provided herein, including, but not limited to those inthe sequence listing, are intended to encompass nucleic acids containingany combination of natural or modified RNA and/or DNA, including, butnot limited to such nucleic acids having modified nucleobases. By way offurther example and without limitation, an oligomeric compound havingthe nucleobase sequence “ATCGATCG” encompasses any oligomeric compoundshaving such nucleobase sequence, whether modified or unmodified,including, but not limited to, such compounds comprising RNA bases, suchas those having sequence “AUCGAUCG” and those having some DNA bases andsome RNA bases such as “AUCGATCG” and oligomeric compounds having othermodified nucleobases, such as “ATmCGAUCG,” wherein ^(m)C indicates acytosine base comprising a methyl group at the 5-position.

Certain compounds described herein (e.g., modified oligonucleotides)have one or more asymmetric center and thus give rise to enantiomers,diastereomers, and other stereoisomeric configurations that may bedefined, in terms of absolute stereochemistry, as (R) or (S), as a or 13such as for sugar anomers, or as (D) or (L), such as for amino acids,etc. Included in the compounds provided herein are all such possibleisomers, including their racemic and optically pure forms, unlessspecified otherwise. Likewise, all cis- and trans-isomers and tautomericforms are also included unless otherwise indicated. Unless otherwiseindicated, compounds described herein are intended to includecorresponding salt forms.

EXAMPLES

The following examples illustrate certain embodiments of the presentdisclosure and are not limiting. Moreover, where specific embodimentsare provided, the inventors have contemplated generic application ofthose specific embodiments. For example, disclosure of anoligonucleotide having a particular motif provides reasonable supportfor additional oligonucleotides having the same or similar motif. And,for example, where a particular high-affinity modification appears at aparticular position, other high-affinity modifications at the sameposition are considered suitable, unless otherwise indicated.

Example 1: Effects of Duplexes Comprising a Lipophilic Conjugate GroupIn Vivo

Duplexes, each consisting of two oligomeric compounds, are described inthe table below. One oligomeric compound of each duplex comprises anantisense oligonucleotide (Isis No. 626112) that is complementary toboth human and mouse Metastasis Associated Lung AdenocarcinomaTranscript 1 (MALAT-1) transcripts. The other oligomeric compound ofeach duplex comprises an oligonucleotide and a lipophilic conjugategroup. The effects of the duplexes on MALAT-1 expression were tested invivo. Wild type C57bl/6 mice each received an intravenous injection, viathe tail vein, of a duplex listed in the table below, Isis No. 626112alone as a control, or saline vehicle alone. Each injection contained100 mg/kg of the antisense oligonucleotide (Isis No. 626112). Eachtreatment group consisted of four mice. Eight days after the injection,the animals were sacrificed. MALAT-1 RNA expression was analyzed inliver, kidney, lung, trigeminal ganglia, frontal cortex, and spinal cordby RT-qPCR and normalized to total RNA using RiboGreen (Thermo FisherScientific, Carlsbad, Calif.). The average results for each group areshown below as the percent normalized MALAT-1 RNA levels relative toaverage results for the vehicle treated animals.

TABLE 1 MALAT-1 expression in vivo MALAT-1 RNA level (% Vehicle) SEQ Fr.Sp. ID Duplex Isis No. Sequence (5′ to 3′) Liver Kidney Lung GangliaCor. Cord NO. n/a 626112G_(es) ^(m)C_(eo) ^(m)C_(eo) A_(eo) G_(eo) G_(ds) 26 67 62 65 101 104 1^(m)C_(ds) T_(ds) G_(ds) G_(ds) T_(ds) T_(ds) A_(ds)T_(ds) G_(ds) A_(eo) ^(m)C_(eo) T_(es) ^(m)C_(es) A_(e) 1 626112G_(es) ^(m)C_(eo) ^(m)C_(eo) A_(eo) G_(eo) G_(ds) 59 59 89 55 119 110 1^(m)C_(ds) T_(ds) G_(ds) G_(ds) T_(ds) T_(ds) A_(ds)T_(ds) G_(ds) A_(eo) ^(m)C_(eo) T_(es) ^(m)C_(es) A_(e) 719228Toco-TEG-U_(ms) G_(ms) A_(ro) 2G_(ro) U_(ro) C_(ro) A_(ro) U_(ro) A_(ro) A_(ro)C_(ro) C_(ro) A_(ro) G_(ro) C_(ro) C_(ro) U_(ro) G_(rs) G_(ms) C_(m) 2626112 G_(es) ^(m)C_(eo) ^(m)C_(eo) A_(eo) G_(eo) G_(ds) 38 39 76 47 10788 1 ^(m)C_(ds) T_(ds) G_(ds) G_(ds) T_(ds) T_(ds) A_(ds)T_(ds) G_(ds) A_(eo) ^(m)C_(eo) T_(es) ^(m)C_(es) A_(e) 719232C10-TEG-U_(ms) G_(ms) A_(ro) G_(ro) 2U_(ro) C_(ro) A_(ro) U_(ro) A_(ro) A_(ro) C_(ro)C_(ro) A_(ro) G_(ro) C_(ro) C_(ro) U_(ro) G_(rs) G_(ms) C_(m) 3 626112G_(es) ^(m)C_(eo) ^(m)C_(eo) A_(eo) G_(eo) G_(ds) 27 63 86 38 120 75 1^(m)C_(ds) T_(ds) G_(ds) G_(ds) T_(ds) T_(ds) A_(ds)T_(ds) G_(ds) A_(eo) ^(m)C_(eo) T_(es) ^(m)C_(es) A_(e) 719233C16-TEG-U_(ms) G_(ms) A_(ro) G_(ro) 2U_(ro) C_(ro) A_(ro) U_(ro) A_(ro) A_(ro) C_(ro)C_(ro) A_(ro) G_(ro) C_(ro) C_(ro) U_(ro) G_(rs) G_(ms) C_(m) 4 626112G_(es) ^(m)C_(eo) ^(m)C_(eo) A_(eo) G_(eo) G_(ds) 18 29 22 21 92 51 1^(m)C_(ds) T_(ds) G_(ds) G_(ds) T_(ds) T_(ds) A_(ds)T_(ds) G_(ds) A_(eo) ^(m)C_(eo) T_(es) ^(m)C_(es) A_(e) 719234Chol-TEG-U_(ms) G_(ms) A_(ro) 2G_(ro) U_(ro) C_(ro) A_(ro) U_(ro) A_(ro) A_(ro)C_(ro) C_(ro) A_(ro) G_(ro) C_(ro) C_(ro) U_(ro) G_(rs) G_(ms) C_(m)Subscripts in the table above: “s” represents a phosphorothioateinternucleoside linkage, “o” represents a phosphate internucleosidelinkage, “d” represents a 2′-deoxynucleoside, “e” represents a 2′-MOEmodified nucleoside, “r” represents a 2′-ribonucleoside (2′-hydroxy),“m” represents 2′-O-methyl modified nucleoside. Superscripts: “m” beforea C represents a 5-methylcysteine. The structures of “Chol-TEG-”,“Toco-TEG-”, “C10-TEG-”, and “C16-TEG-” are shown below.

wherein n is 1 in “C10-TEG-”, and n is 7 in “C16-TEG-”.

Example 2: Effects of Duplexes Comprising a Lipophilic Conjugate GroupIn Vivo

Duplexes, each consisting of two oligomeric compounds, are described inthe table below. One oligomeric compound of each duplex comprises anantisense oligonucleotide (Isis No. 626112 or Isis No. 556089) that iscomplementary to both human and mouse MALAT-1 transcripts. The otheroligomeric compound of each duplex comprises an oligonucleotide and alipophilic conjugate group. The effects of the duplexes on MALAT-1expression were tested in vivo. Wild type C57bl/6 mice each received anintravenous injection, via the tail vein, of a duplex listed in thetable below, Isis No. 626112 alone, Isis No. 556089 alone, or saline.The dosages listed in the table below indicate the amount of Isis No.626112 or Isis No. 556089 that was administered in each injection. Eachtreatment group consisted of three or four mice. Three days after theinjection, the animals were sacrificed. MALAT-1 RNA expression wasanalyzed in heart, macrophages (Macs), trigeminal ganglia (TG), sciaticnerve (SN), and dorsal root ganglion (DRG) by RT-qPCR and normalized tototal RNA using RiboGreen (Thermo Fisher Scientific, Carlsbad, Calif.).The average results for each group are shown below as the percentnormalized MALAT-1 RNA levels relative to average results for thevehicle treated animals.

TABLE 2 MALAT-1 expression in vivo MALAT-1 RNA level SEQ Dosage(% Vehicle) ID Duplex Isis No. Sequence (5′ to 3′) (μmol/kg) Heart MacsTG SN DRG NO. n/a 626112G_(es) ^(m)C_(eo) ^(m)C_(eo) A_(eo) G_(eo) G_(ds) ^(m)C_(ds) T_(ds) 1448 69 45 67 79 1G_(ds) G_(ds) T_(ds) T_(ds) A_(ds) T_(ds) G_(ds) A_(eo) ^(m)C_(eo)T_(es) ^(m)C_(es) A_(e) 4 626112 See above 14 17 16 32 31 44 1 719234Chol-TEG-U_(ms) G_(ms) A_(ro) G_(ro) U_(ro)C_(ro) A_(ro) U_(ro) A_(ro) A_(ro) C_(ro) C_(ro) A_(ro) G_(ro) 2C_(ro) C_(ro) U_(ro) G_(rs) G_(ms) C_(m) n/a 556089G_(ks) ^(m)C_(ks) A_(ks) T_(ds) T_(ds) ^(m)C_(ds) T_(ds) A_(ds) 4.5 6748 79 77 88 3A_(ds) T_(ds) A_(ds) G_(ds) ^(m)C_(ds) A_(ks) G_(ks) ^(m)C_(k) 5 556089See above 4.5 67 40 51 64 59 3 827936Toco-TEG-G_(es) ^(m)C_(es) T_(eo) G_(do) ^(m)C_(do) 4T_(do) A_(do) T_(do) T_(do) A_(do) G_(do) A_(do) A_(do)T_(es) G_(es) ^(m)C_(e) 6 556089 See above 4.5 56 53 64 81 41 3 827937Toco-G_(ms) C_(ms) U_(ro) G_(ro) C_(ro) U_(ro) A_(ro) 5U_(ro) U_(ro) A_(ro) G_(ro) A_(ro) A_(ro) U_(rs) G_(ms) C_(m)See legend for Table 1 for subscripts and superscript key. Subscript “k”represents a cEt modified bicyclic sugar moiety. The structures of“Chol-TEG-” and “Toco-TEG-”, are shown in Example 1. The structure of“Toco-” is:

Example 3: Effects of Duplexes Comprising a Lipophilic Conjugate GroupIn Vivo

A duplex, consisting of two oligomeric compounds, is described in thetable below. One oligomeric compound of the duplex comprises anantisense oligonucleotide (Isis No. 486178) that is complementary toboth human and mouse dystrophia myotonica-protein kinase (DMPK)transcripts. The other oligomeric compound of the duplex comprises anoligonucleotide and a lipophilic conjugate group. The effects of theduplex on DMPK expression were tested in vivo. Wild type Balb-C miceeach received an intravenous injection of the duplex listed in the tablebelow, Isis No. 486178 alone, or saline once per week for four weeks.The dosages listed in the table below indicate the amount of Isis No.486178 that was administered in each injection. Each treatment groupconsisted of three or four mice. Seven days after the final injection,the animals were sacrificed. DMPK mRNA expression was analyzed in liver,diaphragm (Dia), quadriceps (Quad), tibialis anterior (TA), heart, andgastrocnemius (Gast) using RT-qPCR and normalized to total RNA usingRiboGreen (Thermo Fisher Scientific, Carlsbad, Calif.). The averageresults for each group are shown below as the percent normalized DMPKRNA levels relative to average results for the vehicle treated animals.

TABLE 3 DMPK expression in vivo SEQ Dosage DMPK mRNA level (% Vehicle)ID Isis No. Sequence (5′ to 3′) Duplex (mg/kg) Liver Dia Quad TA HeartGast NO. 486178 A_(ks) ^(m)C_(ks) A_(ks) A_(ds) T_(ds) A_(ds) A_(ds) n/a25 8 41 32 26 55 32 6 A_(ds) T_(ds) A_(ds) ^(m)C_(ds) ^(m)C_(ds) G_(ds)50 6 30 13 21 21 18 A_(ks) G_(ks) G_(k) 486178 See above 7 25 5 27 8 1522 18 6 694790 Toco-C_(ms) C_(ms) U_(ro) C_(ro) G_(ro) 50 5 14 8 16 1013 7 G_(ro) U_(ro) A_(ro) U_(ro) U_(ro) U_(ro) A_(ro)U_(ro) U_(rs) G_(ms) U_(m)See legends of Table 1 and 2 for subscripts and superscripts key. Thestructure of “Toco-” is shown in Example 2.

Example 4: Effects of Duplexes Comprising a Lipophilic Conjugate GroupIn Vivo

Duplexes, consisting of two oligomeric compounds, are described in thetable below. One oligomeric compound of each duplex comprises anantisense oligonucleotide (Isis No. 440762) that is complementary tomouse SCARB1 scavenger receptor class B, member 1 (SRB-1) transcript.The other oligomeric compound of the duplex comprises an oligonucleotideand a lipophilic conjugate group. The effects of the duplex on SRB-1expression were tested in vivo. Wild type mice each received anintravenous injection of a duplex listed in the table below, Isis No.440762 alone, or saline once per. The dosages listed in the table belowindicate the amount of Isis No. 440762 that was administered in eachinjection. Each treatment group consisted of three or four mice. Afterthe final injection, the animals were sacrificed. SRB-1 mRNA expressionwas analyzed in liver using RT-qPCR and normalized to total RNA usingRiboGreen (Thermo Fisher Scientific, Carlsbad, Calif.). The averageresults for each group are shown below as the percent normalized SRB-1RNA levels relative to average results for the vehicle treated animals.

TABLE 4 SRB-1 expression in vivo SRB-1 mRNA SEQ Dosage level (% IDIsis No. Sequence (5′ to 3′) Duplex (mg/kg)  Vehicle) NO. 440762T_(ks) ^(m)C_(ks) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds)n/a 3 51 8 A_(ds) ^(m)C_(ds) T_(ds) T_(ks) ^(m)C_(k) 440762 See above 81 33 8 655462 Toco-G_(ms) A_(ms) A_(ro) G_(ro) U_(ro) C_(ro) A_(ro) 9U_(ro) G_(ro) A_(ro) C_(ro) U_(rs) G_(ms) A_(m) 440762 See above 9 1 508 663429 Toco-G_(eo) A_(eo) A_(do) G_(do) T_(eo) ^(m)C_(do) A_(eo) 10T_(do) G_(eo) A_(do) ^(m)C_(eo) T_(do) G_(eo) A_(e) 440762 See above 101 51 8 663430 Toco-G_(es) A_(es) A_(do) G_(do) T_(eo) ^(m)C_(do) A_(eo)10 T_(do) G_(eo) A_(do) ^(m)C_(eo) T_(ds) G_(es) A_(e) 440762 See above11 1 97 8 663752TOCO-G_(fs) A_(fs) A_(fo) G_(fo) U_(fo) C_(fo) A_(fo) U_(fo) 9G_(fo) A_(fo) C_(fo) U_(fs) Gfs A_(f) 440762 See above 12 1 34 8 671663Toco-G_(ms) A_(ms) A_(ro) G_(fo) U_(ro) C_(ro) A_(fo) 9U_(ro) G_(ro) A_(fo) C_(ro) U_(rs) G_(ms) A_(m) 440762 See above 13 1 308 671221 Toco-G_(ks) A_(ks) A_(ro) G_(ro) U_(ro) C_(ro) A_(ro) 9U_(ro) G_(ro) A_(ro) C_(ro) U_(rs) G_(ks) A_(k) 440762 See above 14 1 448 674021 Toco-G_(ms) A_(ms) A_(do) G_(do) T_(do) C_(do) A_(do) 10T_(do) G_(do) A_(do) C_(do) T_(ds) G_(ms) A_(m) 440762 See above 15 1 368 675421 Toco-G_(es) A_(es) A_(ro) G_(ro) U_(ro) C_(ro) A_(ro) U_(ro) 9G_(ro) A_(ro) C_(ro) U_(rs) G_(es) A_(e)See legends of Table 1 and 2 for subscripts and superscripts key.Subscript “f” indicates a 2′-fluoro modification. The structure of“Toco-” is shown in Example 2.

1-377. (canceled)
 378. A duplex comprising a first oligomeric compoundand a second oligomeric compound wherein: the first oligomeric compoundcomprises a first modified oligonucleotide consisting of 10-30 linkednucleosides, having a nucleobase sequence that is at least 80%complementary to the nucleobase sequence of the second oligomericcompound and that is at least 80% complementary to an extra-hepaticnucleic acid target; wherein neither the first oligomeric compound northe duplex is an RNAi compound; the second oligomeric compound comprisesa conjugate group and a second modified oligonucleotide consisting of10-30 linked nucleosides; wherein the conjugate group comprises aconjugate moiety and a conjugate linker, wherein the conjugate moiety ischolesterol; and wherein the conjugate linker comprises at least onecleavable moiety; and wherein the extra-hepatic nucleic acid target isexpressed in the brain or spinal cord.
 379. The duplex of claim 378,wherein the first modified oligonucleotide comprises at least onemodified nucleoside.
 380. The duplex of claim 379, wherein the at leastone modified nucleoside of the first modified oligonucleotide comprisesa bicyclic sugar moiety.
 381. The duplex of claim 380, wherein at leastone bicyclic sugar moiety comprises a 2′-4′ bridge, wherein the 2′-4′bridge is selected from —O—CH₂—; and —O—CH(CH₃)—.
 382. The duplex ofclaim 379, wherein the at least one modified nucleoside of the firstmodified oligonucleotide comprises a non-bicyclic sugar modification.383. The duplex of claim 382, wherein at least one non-bicyclic sugarmodification is selected from 2′-MOE and 2′-OMe.
 384. The duplex ofclaim 378, wherein the first modified oligonucleotide comprises a sugarmotif having: a 5′-region consisting of 1-5 linked 5′-nucleosides; acentral region consisting of 6-10 linked central region nucleosides; anda 3′-region consisting of 1-5 linked 3′-region nucleosides; wherein thenucleosides of the 5′-region, the 3′-region, and the central region arecontiguous, and the central region nucleosides each comprise anunmodified DNA sugar moiety.
 385. The duplex of claim 384, wherein eachof the 5′-region nucleosides and each of the 3′-region nucleosidescomprise a modified sugar moiety.
 386. The duplex of claim 378, whereinthe second modified oligonucleotide comprises at least one modifiednucleoside.
 387. The duplex of claim 386, wherein the at least onemodified nucleoside of the first modified oligonucleotide comprises anon-bicyclic sugar modification.
 388. The duplex of claim 387, whereinat least one non-bicyclic sugar modification is selected from 2′-MOE and2′-OMe.
 389. The duplex of claim 378, wherein the second modifiedoligonucleotide comprises a sugar motif having: a 5′-region consistingof 1-5 linked 5′-nucleosides; a central region consisting of 6-10 linkedcentral region nucleosides; and a 3′-region consisting of 1-5 linked3′-region nucleosides; wherein the nucleosides of the 5′-region, the3′-region, and the central region are contiguous, and the central regionnucleosides each comprise an unmodified RNA sugar moiety.
 390. Theduplex of claim 389, wherein each of the 5′-region nucleosides and eachof the 3′-region nucleosides comprise a modified sugar moiety.
 391. Theduplex of claim 378, wherein the first modified oligonucleotidecomprises at least one modified internucleoside linkage.
 392. The duplexof claim 391, wherein the at least one modified internucleoside linkageis a phosphorothioate internucleoside linkage.
 393. The duplex of claim392, wherein each internucleoside linkage of the first modifiedoligonucleotide is a phosphorothioate internucleoside linkage.
 394. Theduplex of claim 392, wherein each internucleoside linkage of the firstmodified oligonucleotide is either an unmodified phosphodiesterinternucleoside linkage or a phosphorothioate internucleoside linkage.395. The duplex of claim 378, wherein the second modifiedoligonucleotide comprises at least one modified internucleoside linkage.396. The duplex of claim 395, wherein the at least one modifiedinternucleoside linkage is a phosphorothioate internucleoside linkage.397. The duplex of claim 396, wherein each internucleoside linkage ofthe second modified oligonucleotide is either an unmodifiedphosphodiester internucleoside linkage or a phosphorothioateinternucleoside linkage.
 398. The duplex of claim 397, wherein 1-3terminal internucleoside linkages of the second modified oligonucleotideare phosphorothioate internucleoside linkages, and each remaininginternucleoside linkage of the second modified oligonucleotide is aphosphodiester internucleoside linkage.
 399. The duplex of claim 378,wherein the first modified oligonucleotide and the second modifiedoligonucleotide are the same length, and each modified oligonucleotideconsists of 12-16, 14-16, 14-18, 16-18, 14-20, 16-20, 18-20, 18-22, 14,16, 18, or 20 linked nucleosides.
 400. The duplex of claim 378, whereinthe cleavable moiety is a phosphate group.
 401. The duplex of claim 378,wherein the extra-hepatic nucleic acid target is not expressed in theliver at a significant level.